An Iso mutant, a genetically engineered strain and its application in synthesizing vanillin

Abstract

The present invention provides an Iso mutant, a genetically engineered strain and its application in the synthesis of vanillin, belonging to the field of genetic engineering technology. The present invention mutates any one or more sites of the 27th, 77th, 91st, 139th, 156th, 227th, 417th, and 434th positions away from the center of the pocket of 4-vinylguaiacol oxygenase Iso, and obtains a genetically engineered strain capable of efficiently catalyzing ferulic acid to generate vanillin by a two-step enzymatic method. The genetically engineered strain can efficiently generate vanillin, and at a substrate concentration of 100mmol / L, the vanillin conversion rate can reach 68.5%-85.73%, and the yield can reach 10.41-13.03g / L, with good industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to an Iso mutant, a genetic engineering strain and application thereof in synthesizing vanillin. Background Art

[0002] Vanillin is one of the most widely used condiments in the world and is in high demand as a flavoring agent in various foods. Vanillin also has antimicrobial and antioxidant properties and is therefore also used as a food preservative or combined with chitosan / polyvinyl alcohol to prepare active films for food packaging. In addition to being widely used as a condiment and masking agent, vanillin is also in great demand in drug production due to its antioxidant, antimicrobial, anti-inflammatory, anti-nociceptive, analgesic and anti-cancer properties. Producing high-value vanillin to meet the rapidly growing demand is very important for the food, pharmaceutical, cosmetic and fine chemical industries.

[0003] Vanillin extracted from vanilla pods is a natural product, but the supply of raw vanilla pods is limited. The extraction method is labor-intensive, has a long processing cycle, and the price of the product is also very expensive, making the vanillin obtained by natural extraction far from meeting market demand. The production of vanillin by chemical synthesis generally has many by-products that are difficult to separate, and the production process can cause environmental pollution. At the same time, compared with natural vanillin, chemically synthesized vanillin has a single flavor and lower quality, so its selling price is also much lower than natural vanillin.

[0004] Due to various shortcomings of natural extraction and chemical synthesis, people pay more and more attention to the use of biological methods to synthesize vanillin, which breaks through the limitations of plant extraction methods, has the advantages of short cycle and no influence of environmental factors, and avoids the shortcomings of chemical synthesis. It has low energy consumption and low pollution, and the obtained product quality is better.

[0005] Harmann et al. used the lipase Chirazyme to catalyze the substrates isoeugenol and coniferyl aldehyde, and obtained vanillin with a mass fraction of 30.4% and 83.1%, respectively. Some researchers tried to add ferulic acid as a substrate when culturing the aerial root tissue of vanilla and obtained 0.7% (w / w) vanillin. However, the above methods have the disadvantage of being difficult to control and have low yields, so they are difficult to apply industrially.

[0006] Vanillin produced by transforming natural raw materials using microorganisms is recognized as natural vanillin. The vanillin obtained by this method meets people's demand for healthy food and can also meet the market's demand for natural vanillin. There are many strains in nature that can transform substances such as ferulic acid, eugenol and isoeugenol into vanillin. By optimizing fermentation and genetic engineering of these strains, a new method for increasing the yield of vanillin can be developed, which has industrial application prospects. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides an Iso mutant, a genetically engineered strain and its application in the synthesis of vanillin. Enzyme substrate channel engineering has become an emerging protein engineering strategy. Molecular modification of amino acid residues near the enzyme substrate binding pocket can change the substrate specificity, regioselectivity, stereoselectivity, etc. of the enzyme, but there are few studies on the modification of sites far from the center of the enzyme pocket. The present invention mutates any one or more sites of the 27th, 77th, 91st, 139th, 156th, 227th, 417th, and 434th positions away from the center of the pocket of 4-vinylguaiacol oxygenase Iso to obtain a genetically engineered strain that can efficiently catalyze ferulic acid to produce vanillin through a two-step enzymatic method. The genetically engineered strain can efficiently produce vanillin, and at a substrate concentration of 100mmol / L, the vanillin conversion rate can reach 68.5-85.73%, and the yield can reach 10.41-13.03g / L, which has good industrial application prospects.

[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 "H49F" represents mutation of histidine H at position 49 to phenylalanine F.

[0012] In the present invention, the term "Y77F" represents mutation of tyrosine Y at position 77 to phenylalanine F.

[0013] In the present invention, the term "K91R" represents mutation of lysine K at position 91 to arginine R.

[0014] In the present invention, the term "G139A" represents mutation of glycine G at position 139 to alanine A.

[0015] In the present invention, the term "Y156D" represents mutation of tyrosine Y at position 156 to aspartic acid D.

[0016] In the present invention, the term "A227V" represents mutation of alanine A at position 227 to valine V.

[0017] In the present invention, the term "P408C" represents the mutation of the proline P at position 408 to the cysteine ​​C.

[0018] In the present invention, the term "H417F" represents the mutation of histidine H at position 417 to phenylalanine F.

[0019] In the present invention, the term "D434Y" represents mutation of aspartic acid D at position 434 to tyrosine Y.

[0020] The technical solution of the present invention is:

[0021] In one aspect, the present invention provides an Iso mutant, wherein the Iso mutant comprises a mutation in a site away from the center of the pocket of 4-vinylguaiacol oxygenase Iso.

[0022] Specifically, the amino acid sequence of the 4-vinylguaiacol oxygenase Iso is shown in SEQ ID NO.1.

[0023] SEQ ID NO.1:

[0024] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPPMMRAQVHGAWVSARQLANARQNVDSEAA.

[0025] Specifically, the site away from the center of the pocket is any one or more of the 27th, 77th, 91st, 139th, 156th, 227th, 417th, and 434th sites of SEQ ID NO.1.

[0026] Preferably, the mutation comprises mutating aspartic acid D at position 27 to proline P.

[0027] Preferably, the mutation comprises mutating tyrosine Y at position 77 to phenylalanine F.

[0028] Preferably, the mutation comprises mutating the lysine K at position 91 to arginine R.

[0029] Preferably, the mutation comprises mutating glycine G at position 139 to alanine A.

[0030] Preferably, the mutation comprises mutating the tyrosine Y at position 156 to aspartic acid D.

[0031] Preferably, the mutation comprises mutating the alanine A at position 227 to valine V.

[0032] Preferably, the mutation comprises mutating the histidine H at position 417 to phenylalanine F.

[0033] Preferably, the mutation comprises mutating aspartic acid D at position 434 to tyrosine Y.

[0034] Preferably, the Iso mutant comprises:

[0035] (1) a mutant D27P in which the aspartic acid D at position 27 is mutated to proline P; or

[0036] (2) mutant Y77F in which the tyrosine Y at position 77 is mutated to phenylalanine F; or

[0037] (3) a mutant K91R in which the lysine K at position 91 is mutated to arginine R; or

[0038] (4) mutant G139A in which the glycine G at position 139 is mutated to alanine A; or

[0039] (5) mutant Y156D in which the tyrosine Y at position 156 is mutated to aspartic acid D; or

[0040] (6) mutant A227V in which alanine A at position 227 is mutated to valine V; or

[0041] (7) a mutant H417F in which the histidine H at position 417 is mutated to phenylalanine F; or

[0042] (8) The mutant D434Y was formed by mutating the aspartic acid D at position 434 to tyrosine Y.

[0043] Further preferably, the Iso mutant has an amino acid sequence as shown in SEQ ID NO.2-9.

[0044] The mutant D27P has the amino acid sequence as shown in SEQ ID NO.2.

[0045] SEQ ID NO.2:

[0046] MPNYPQTIHFVGLNTPVGIEWSARNLPVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0047] The mutant Y77F has the amino acid sequence as described in SEQ ID NO.3.

[0048] SEQ ID NO.3:

[0049] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDFAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0050] The mutant K91R has the amino acid sequence as set forth in SEQ ID NO.4.

[0051] SEQ ID NO.4:

[0052] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLERDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0053] The mutant G139A has the amino acid sequence as set forth in SEQ ID NO.5.

[0054] SEQ ID NO.5:

[0055] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLAYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0056] The mutant Y156D has the amino acid sequence as set forth in SEQ ID NO.6.

[0057] SEQ ID NO.6:

[0058] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYDGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0059] The mutant A227V has the amino acid sequence as set forth in SEQ ID NO.7.

[0060] SEQ ID NO.7:

[0061] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYVIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0062] The mutant H417F has the amino acid sequence as set forth in SEQ ID NO.8.

[0063] SEQ ID NO.8:

[0064] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVFVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0065] The mutant D434Y has the amino acid sequence as set forth in SEQ ID NO.9.

[0066] SEQ ID NO.9:

[0067] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFYSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0068] In some embodiments, the mutant H49F has the amino acid sequence shown in SEQ ID NO. 10.

[0069] SEQ ID NO. 10:

[0070] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAFPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0071] In some embodiments, the mutant P408C has the amino acid sequence shown in SEQ ID NO.11.

[0072] SEQ ID NO.11:

[0073] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLECGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPPMMRAQVHGAWVSARQLANARQNVDSEAA.

[0074] On the other hand, the present invention provides an Iso mutant genetically engineered strain, wherein the Iso mutant genetically engineered strain overexpresses the above-mentioned Iso mutant.

[0075] Specifically, the Iso mutant genetic engineering strain uses Escherichia coli as a host and heterologously overexpresses the Iso mutant.

[0076] Preferably, the Iso mutant genetically engineered strains include BL21-pET28a-Iso D27P, BL21-pET28a-Iso Y77F, BL21-pET28a-Iso K91R, BL21-pET28a-Iso G139A, BL21-pET28a-IsoY156D, BL21-pET28a-Iso A227V, BL21-pET28a-Iso H417F, and BL21-pET28a-Iso D434Y.

[0077] In another aspect, the present invention provides the use of the above-mentioned Iso mutant or the Iso mutant genetically engineered strain in catalyzing the synthesis of vanillin from ferulic acid.

[0078] Specifically, the Iso mutant or the Iso mutant genetically engineered strain catalyzes 4-vinylguaiacol to produce vanillin.

[0079] Specifically, the application also includes the use of ferulic acid decarboxylase Bpf or its genetically engineered strain.

[0080] Preferably, the ferulic acid decarboxylase Bpf has an amino acid sequence as shown in SEQ ID NO.12, and the genetically engineered strain uses Escherichia coli as a host to heterologously overexpress the gene encoding ferulic acid decarboxylase Bpf.

[0081] SEQ ID NO.12:

[0082] MDQFVGLHMIYTYENGWEYEIYIKNDHTIDYRIHSGMVGGRWVRDQEVNIVKLTKGVYKVSWTEPTGTDVSLNFMPEEKRMHGVIFFPKWVHERPDITVCYQNDYIDLMKESREKYETYPKYVVPEFADITYIHHAGVNDETIIAEAPYEGMTDEIRAGRK.

[0083] Further preferably, the genetically engineered strain of ferulic acid decarboxylase Bpf is BL21-pET28a-Bpf.

[0084] Specifically, the ferulic acid decarboxylase Bpf or its genetically engineered strain catalyzes ferulic acid to produce 4-vinylguaiacol.

[0085] In another aspect, the present invention provides a method for synthesizing vanillin, the method comprising the following steps:

[0086] S1, using ferulic acid as substrate, adding crude enzyme solution of ferulic acid decarboxylase Bpf genetic engineering strain for transformation;

[0087] S2. After the transformation is completed, the crude enzyme solution of the Iso mutant genetic engineering strain is added to react to obtain vanillin.

[0088] The beneficial effects of the present invention are:

[0089] The invention mutates any one or more sites of the 27th, 77th, 91st, 139th, 156th, 227th, 417th and 434th positions away from the center of the pocket of 4-vinylguaiacol oxygenase Iso to obtain a genetic engineering strain capable of efficiently catalyzing ferulic acid to generate vanillin through a two-step enzymatic method. The genetic engineering strain can efficiently generate vanillin, with a vanillin conversion rate of 68.5%-85.73% at a substrate concentration of 100 mmol / L, and a yield of 10.41-13.03 g / L, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 This is the HPLC detection spectrum of ferulic acid, vanillin and 4-vinylguaiacol.

[0091] 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.

[0092] 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.

[0093] Figure 4 It 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 mutant D27P; 3 represents lane 3, which is mutant H49F; 4 represents lane 4, which is mutant Y77F; 5 represents lane 5, which is mutant K91R; 6 represents lane 6, which is mutant G139A; 7 represents lane 7, which is mutant Y156D; 8 represents lane 8, which is mutant A227V; 9 represents lane 9, which is mutant P408C; 10 represents lane 10, which is mutant H417F; 11 represents lane 11, which is mutant D434Y. DETAILED DESCRIPTION

[0094] 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.

[0095] The culture medium formula involved in the present invention is as follows:

[0096] 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.

[0097] 2. LB solid medium (1 L): Add 15 g agar to the LB liquid medium formula.

[0098] 3. 2YT medium (1L): 16g tryptone, 10g yeast extract, 5g sodium chloride, dilute to 1L with pure water, and sterilize with high-pressure steam at 121℃ for 15min.

[0099] 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.

[0100] 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.

[0101] Basic Experimental Example 1 Establishment of Vanillin Detection Method

[0102] 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).

[0103] 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.

[0104] 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.

[0105] Basic Experimental Example 2 Determination of Enzyme Activity of Vinylguaiacol Oxygenase

[0106] 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.

[0107] 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 min is defined as one unit U.

[0108] Example 1 Construction of pET28a-Bpf, a gene expression vector related to the vanillin synthesis pathway

[0109] 1.1. Construction of pET28a-Bpf vector

[0110] The ferulic acid decarboxylase Bpf (NCBI accession number ACO50701.1) from Bacillus has an amino acid sequence as shown in SEQ ID NO. 12. The codons of Escherichia coli were optimized, and the codon-optimized sequence was sent to General Biotechnology (Anhui) Co., Ltd. for synthesis. The primers P1 (Bpf-F, Bpf-R) were used for PCR amplification (PrimeSTAR GXLDNA Polymerase, Takara Code No. R050A), and a linear fragment Bpf (483 bp) was amplified, having a nucleotide sequence as shown in SEQ ID NO. 13.

[0111] SEQ ID NO.13:

[0112] ATGGACCAGTTCGTTGGTCTGCACATGATCTACACCTACGAAAACGGTTGGGAATACGAAATCTACATCAAAAACGATCACACCATCGACTACCGTATCCACTCTGGTATGGTTGGTGGTCGTTGGGTTCGCGATCAGGAAGTTAACATCGTTAAACTGACCAAAGGTGTTTACAAAGTTTCCTGGACCGAACCGACCGGCACCGACGTTTCTCTCAACTTCATGCCAGAAGAAAAACGTAT GCACGGTGTTATCTTCTTCCCGAAGTGGGTTCACGAACGTCCAGACATTACCGTTTGCTACCAGAACGACTACATTGACCTGATGAAAGAATCTCGTGAAAAATACGAGACCTACCCGAAATACGTTGTTCCAGAGTTCGCGGACATCACCTACATCCACCACGCGGGTGTTAACGACGAAACCATCATCGCGGAAGCGCCGTACGAAGGTATGACCGACGAGATCCGTGCGGGTCGTAAA.

[0113] When amplifying the linearized pET28a vector, the nucleotide sequence of pET28a (as shown in SEQ ID NO. 14) was amplified by PCR using primers P2 (pET28a-F, pET28a-R) to obtain the linearized pET28a vector (5308 bp).

[0114] SEQ ID NO.14:

[0115]

[0116] The primer sequences used are shown in Table 1:

[0117] Table 1 Primer P1 and Primer P2 sequence information

[0118]

[0119] In Table 1, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0120] 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).

[0121] 1.2. Cultivation and induced expression of recombinant strains

[0122] 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.

[0123] Take 50OD Ecoli-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.

[0124] 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.

[0125] Example 2 Construction of pET28a-Iso, an expression vector for genes related to the synthesis pathway of vanillin

[0126] 2.1. Construction of pET28a-Iso vector

[0127] Using the BLAST tool, all amino acid sequences with a similarity of more than 30% to the Cso2 sequence were searched in the Uniprot database (the amino acid length was set between 450-550), all similar sequences were constructed by the AlphaFold2-based structural modeling method, and then a structure-based evolutionary tree was constructed, and 5 enzymes with certain representativeness were selected 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.

[0128] 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 1.1 of Example 1. 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. 19.

[0129] SEQ ID NO.19:

[0130]

[0131] The primer sequences used are shown in Table 2:

[0132] Table 2 Primer P3 sequence information

[0133]

[0134] In Table 2, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0135] 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).

[0136] 2.2. Cultivation and induced expression of recombinant strains

[0137] The protein expression and detection steps of the recombinant strain BL21-pET28a-Iso were the same as those described in 1.2 of Example 1. The expression of the recombinant strain BL21-pET28a-Iso enzyme was as follows: 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.

[0138] Example 3 Construction of 4-vinylguaiacol oxygenase mutants based on whole plasmid PCR

[0139] Using the recombinant plasmid pET28a-Iso (Example 2) as a template, PCR (PrimeSTAR HSDNA Polymerase, Takara Code No. R010A) was used for in vitro mutation amplification. The primer sequences used for site-directed mutagenesis are shown in Table 3 below.

[0140] Table 3 Primer sequence information for site-directed mutagenesis

[0141]

[0142]

[0143] In Table 3, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0144] After PCR, 10 μL of PCR product was added to 1 μL of restriction endonuclease DpnⅠ to digest the template plasmid. After reacting at 37°C for 2 h, the digested product was transferred into E. coli BL21 (DE3) (Weidi Biotechnology, CAT#: EE1002) according to the method described in 1.2 of Example 1, spread on LB solid plate (containing 50 μg / mL Kana), and cultured at 37°C overnight. Four transformants were picked and cultured in LB liquid medium (containing 50 μg / mL Kana) for 6 h before being sent for sequencing. Correctly sequenced BL21-pET28a-IsoD27P, BL21-pET28a-Iso H49F, BL21-pET28a-Iso Y77F, BL21-pET28a-Iso K91R, BL21-pET28a-Iso G139A, and BL21-pET28a-Iso were obtained. Y156D, BL21-pET28a-Iso A227V, BL21-pET28a-IsoP408C, BL21-pET28a-Iso H417F, BL21-pET28a-Iso D434Y.

[0145] Example 4 Protein expression and purification of 4-vinylguaiacol oxygenase and its mutants

[0146] 4.1 Protein Expression

[0147] 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.

[0148] 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.

[0149] 4.2 Protein Purification

[0150] 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.

[0151] 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, lane 3 represents mutant H49F, lane 4 represents mutant Y77F, lane 5 represents mutant K91R, lane 6 represents mutant G139A, lane 7 represents mutant Y156D, lane 8 represents mutant A227V, lane 9 represents mutant P408C, lane 10 represents mutant H417F, and lane 11 represents mutant D434Y.

[0152] Example 5 Comparison of the activities of different 4-vinylguaiacol oxygenase mutants and the parent enzyme

[0153] 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, H49F, Y77F, K91R, G139A, Y156D, A227V, P408C, H417F, and D434Y were 5.48U / mg, 2.50U / mg, 3.18U / mg, 3.07U / mg, 3.20U / mg, 5.16U / mg, 3.55U / mg, 2.42U / mg, 4.09U / mg, and 3.64U / mg, respectively. The results are shown in Table 4.

[0154] Table 4 Comparison of the activities of different 4-vinylguaiacol oxygenase mutants and the parent enzyme

[0155] Enzymes Specific activity U / mg Iso 2.80 D27P 5.48 H49F 2.50 Y77F 3.18 K91R 3.07 G139A 3.20 Y156D 5.16 A227V 3.55 P408C 2.42 H417F 4.09 D434Y 3.64

[0156] Example 5 Comparison of the conversion rate and yield of vanillin synthesized by 4-vinylguaiacol oxygenase mutant and parent crude enzyme

[0157] 5.1. Strain culture and crude enzyme solution preparation

[0158] The strain culture and crude enzyme solution preparation refer to 1.2 described in Example 1.

[0159] 5.2. Conversion of Ferulic Acid to Vanillin

[0160] 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.

[0161] 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.

[0162] The results showed that after 5 h of reaction, the vanillin conversion rate of the parent Iso was 65.53% and the yield was 9.66 g / L. The conversion rates of mutants D27P, H49F, Y77F, K91R, G139A, Y156D, A227V, P408C, H417F, and D434Y were 85.73%, 53.85%, 71.33%, 68.5%, 71.21%, 83.81%, 74.24%, 51.4%, 79.33%, and 76.75%, respectively; and the yields were 13.03 g / L, 8.19 g / L, 10.84 g / L, 10.41 g / L, 10.82 g / L, 12.74 g / L, 11.28 g / L, 7.81 g / L, 12.05 g / L, and 11.67 g / L, respectively. The results are shown in Table 5.

[0163] Table 5 Comparison of conversion rate and yield

[0164] Enzymes Conversion Rate % Yield g / L Iso 63.53％ 9.66 D27P 85.73％ 13.03 H49F 53.85％ 8.19 Y77F 71.33％ 10.84 K91R 68.50％ 10.41 G139A 71.21％ 10.82 Y156D 83.81％ 12.74 A227V 74.24％ 11.28 P408C 51.40％ 7.81 H417F 79.33％ 12.05 D434Y 76.75％ 11.67

[0165] 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. An Iso mutant, characterized in that The Iso mutant is a mutation of a site away from the center of the pocket of 4-vinylguaiacol oxygenase Iso; The amino acid sequence of the 4-vinylguaiacol oxygenase Iso is shown in SEQ ID NO.1, and the site away from the center of the pocket is the 27th position of SEQ ID NO.1; the amino acid sequence of the Iso mutant is shown in SEQ ID NO.

2.

2. An Iso mutant genetically engineered strain, characterized in that: The Iso mutant genetically engineered strain overexpresses the Iso mutant according to claim 1.

3. The Iso mutant genetically engineered strain according to claim 2, characterized in that: The Iso mutant genetic engineering strain uses Escherichia coli as a host and heterologously over-expresses the Iso mutant.

4. Use of the Iso mutant according to claim 1 or the Iso mutant genetically engineered strain according to any one of claims 2 to 3 in catalyzing the synthesis of vanillin from ferulic acid, characterized in that: The application includes using ferulic acid decarboxylase Bpf or its genetically engineered strain to catalyze ferulic acid to produce 4-vinylguaiacol; the Iso mutant or the Iso mutant genetically engineered strain catalyzes 4-vinylguaiacol to produce vanillin; The ferulic acid decarboxylase Bpf has an amino acid sequence as shown in SEQ ID NO.

12. The genetically engineered strain uses Escherichia coli as a host and heterologously overexpresses a gene encoding the ferulic acid decarboxylase Bpf.

5. A method for synthesizing vanillin, characterized in that: The method comprises the following steps: S1, using ferulic acid as substrate, adding crude enzyme solution of ferulic acid decarboxylase Bpf genetic engineering strain for transformation; S2. After the transformation is completed, the crude enzyme solution of the Iso mutant genetically engineered strain according to any one of claims 2 to 3 is added to react to obtain vanillin.

Images