Iso mutant and its genetic engineering bacteria and application

Abstract

The present invention provides an Iso mutant and its genetically engineered bacteria and application, belonging to the field of genetic engineering technology. The present invention mutates the pocket center site of 4-vinylguaiacol oxygenase Iso, and the enzyme activity of the mutant M351F is 1.93 times that before the mutation. At a substrate concentration of 100mmol / L, the vanillin conversion rate is 84.72%, and the yield reaches 12.88g / L. The Iso mutant provided by the present invention can efficiently catalyze 4-vinylguaiacol to generate vanillin, and has good industrial application prospects.

Description

[0001] The present invention is a divisional application of a Chinese patent filed with the China Patent Office on May 23, 2024, with application number 202410652461.0 and invention name “4-vinylguaiacol oxygenase mutants and genetically engineered bacteria and their applications”, all contents of which are incorporated by reference in the application. Technical Field

[0002] The invention belongs to the field of genetic engineering, and specifically relates to an Iso mutant and genetically engineered bacteria and applications thereof. Background Art

[0003] Vanillin, also known as vanillin, has a chemical name of 3-methoxy-4-hydroxybenzaldehyde. Its molecular formula is C8H8O3, and its relative molecular weight is 152. Pure vanillin is white or light yellow powder or needle-shaped crystals, with the aroma of vanilla beans and strong milky aroma. Vanillin is known as the "Queen of Spice" for its unique aroma characteristics that cannot be artificially compounded and its wide range of applications. It is widely used in food, medicine, feed, condiments, and cosmetics industries.

[0004] Currently, the vanillin on the market includes natural vanillin and synthetic vanillin: natural vanillin extracted from plants has a long plant planting cycle and a complex vanillin extraction process, and its output is extremely limited and expensive, which can only provide less than 1% of the annual market demand. Compared with natural vanillin, synthetic vanillin obtained by chemical methods has a large output and a very low price, so synthetic vanillin occupies the vast majority of the vanillin seasoning market. However, synthetic vanillin produced by chemical processes has more by-products, lower product quality and is prone to adverse effects on human health, and its use in the food and pharmaceutical industries is strictly restricted.

[0005] Microbial transformation method uses natural substances as substrates and adopts biological transformation methods, so the vanillin obtained is considered to be "natural vanillin". Various microorganisms in nature, such as Streptomyces, Bacillus, Pseudomonas, Pycnoporus vermilionii, Enterobacter, etc., can transform different substrates to synthesize vanillin. Through the discovery and identification of related enzymes and genes, as well as the study of metabolic pathways and fermentation conditions, it provides more options and opportunities for genetic engineering to increase vanillin production and large-scale production of vanillin through microbial transformation.

[0006] Patent CN106754802A mutated the amino acids of isoeugenol monooxygenase from Pseudomonas putida IE27 in an effort to increase the activity of the enzyme, but the activity was only increased by 2 times, and the maximum vanillin concentration was less than 1.2 g / L.

[0007] Therefore, developing a method for producing vanillin that is safe, has high enzyme activity, high conversion efficiency, and is suitable for large-scale production is a current research hotspot. Summary of the invention

[0008] In view of the above shortcomings, the present invention provides an Iso mutant and its genetically engineered bacteria and application. The present invention mutates the pocket center site of 4-vinylguaiacol oxygenase Iso, and the enzyme activity of the mutant M351F is 1.93 times that before the mutation. At a substrate concentration of 100mmol / L, the vanillin conversion rate is 84.72%, and the yield reaches 12.88g / L. The Iso mutant provided by the present invention can efficiently catalyze 4-vinylguaiacol to produce vanillin, and has good industrial application prospects.

[0009]

[0010] the term:

[0011] In the present invention, the term "V58W" represents mutation of valine V at position 58 to tryptophan W.

[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 "T216L" represents mutation of threonine T at position 216 to leucine L.

[0014] In the present invention, the term "S280M" represents mutation of serine S at position 280 to methionine M.

[0015] In the present invention, the term "G307P" represents mutation of glycine G at position 307 to proline P.

[0016] In the present invention, the term "M351F" represents mutation of methionine M at position 351 to phenylalanine F.

[0017] The technical solution of the present invention is:

[0018] On the one hand, the present invention provides a 4-vinylguaiacol oxygenase mutant, characterized in that the 4-vinylguaiacol oxygenase mutant comprises a mutation in the central site 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.

[0019] SEQ ID NO.1:

[0020] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPPMMRAQVHGAWVSARQLANARQNVDSEAA.

[0021] Specifically, the pocket center site is any one or more of the 216th, 280th, 307th, and 351st sites of SEQ ID NO.1.

[0022] More specifically, the mutation includes mutating the threonine T at position 216 to methionine M.

[0023] More specifically, the mutation includes mutating the threonine T at position 216 to leucine L.

[0024] More specifically, the mutation includes mutating the serine S at position 280 to methionine M.

[0025] More specifically, the mutation includes mutating the glycine G at position 307 to proline P.

[0026] More specifically, the mutation includes mutating the methionine M at position 351 to phenylalanine F.

[0027] Preferably, the 4-vinylguaiacol oxygenase mutant comprises:

[0028] (1) a mutant T216M in which the threonine T at position 216 is mutated to methionine M; or

[0029] (2) a mutant T216L in which the threonine T at position 216 is mutated to leucine L; or

[0030] (3) a mutant S280M in which the serine S at position 280 is mutated to methionine M; or

[0031] (4) a mutant G307P in which the glycine G at position 307 is mutated to proline P; or

[0032] (5) A mutant M351F in which the methionine M at position 351 was mutated to phenylalanine F.

[0033] Further preferably, the 4-vinylguaiacol oxygenase mutant comprises an amino acid sequence as shown in SEQ ID NO. 2-6.

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

[0035] SEQ ID NO.2:

[0036] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSMIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPPMPMRAQVHGAWVSARQLANARQNVDSEAA.

[0037] The mutant T216L has an amino acid sequence as shown in SEQ ID NO.3.

[0038] SEQ ID NO.3:

[0039] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSLIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0040] The mutant S280M has the amino acid sequence shown in SEQ ID NO.4.

[0041] SEQ ID NO.4:

[0042] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVMAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0043] The mutant G307P has the amino acid sequence shown in SEQ ID NO.5.

[0044] SEQ ID NO.5:

[0045] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFPFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0046] The mutant M351F has an amino acid sequence as shown in SEQ ID NO.6.

[0047] SEQ ID NO.6:

[0048] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIVLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTPVWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAHWAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDFPRLRDADQGRPYRAAWYLSMNPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPMPMPMRAQVHGAWVSARQLANARQNVDSEAA。

[0049] In some embodiments, the mutant V58W has the amino acid sequence shown in SEQ ID NO.7.

[0050] SEQ ID NO.7:

[0051] MPNYPQTIHFVGLNTPVGIEWSARNLDVIGTIPAEIDGAFFRAVPDPAHPPMFDDDIWLSGDGMVAKFAIHDGKVDYAIRYVETERYRLEKDARRALFGQYRNPFTDDASVAGRDRTVANTTP VWHGGRLLMTKEDGLGYQVDPDTLETLGRFDYYGALKSQTFTAHPRVDPETGEMFFFGYEADGLCSTKISYAIADRDGKLISEQWFDQPYCSTIHDFAITEHYAIFPIFPTTADLDRLKAGGAH WAHQQDLESWVGIMPRYGKVEEMRWFKGRNGVSAFHLVNAYEEDGFVHLDLCLSDTNAFGFMREAGGIQRDQRDIQGGLTRWTFDLSKEGDMFEERLVGPPGDMPRLRDADQGRPYRAAWYLSM NPQGGPPLPGGPVGVAFNALLRIEPGNGRIDMMGLEPGMAISEPVHVVPSTQDEHEGWLLMVVDRQAGEADFKSELWVVEAGAIAAGPIARVPPMPMRAQVHGAWVSARQLANARQNVDSEAA.

[0052] In another aspect, the present invention provides a coding gene for encoding the 4-vinylguaiacol oxygenase mutant as claimed in claim 1.

[0053] In yet another aspect, the present invention provides a recombinant vector carrying the above-mentioned coding gene.

[0054] In yet another aspect, the present invention provides a genetically engineered strain, comprising the above-mentioned recombinant vector, and heterologously overexpressing a 4-vinylguaiacol oxygenase mutant.

[0055] Specifically, the genetically engineered strain uses the recombinant plasmid pET28a-Iso as a template, performs in vitro mutation amplification using primers, and then is transformed into Escherichia coli.

[0056] Preferably, the genetically engineered strain comprises BL21-pET28a-Iso T216M, BL21-pET28a-IsoT216L, BL21-pET28a-Iso S280M, BL21-pET28a-Iso G307P or BL21-pET28a-Iso M351F.

[0057] In another aspect, the present invention provides a genetically engineered preparation, which comprises one or more of the culture, culture extract, cell disruptor, bacterial body, fermentation broth, fermentation broth precipitate, and lyophilized powder of the above-mentioned genetically engineered strain.

[0058] In another aspect, the present invention provides the use of the above-mentioned 4-vinylguaiacol oxygenase mutant, the above-mentioned encoding gene, the above-mentioned recombinant vector, the above-mentioned genetically engineered strain or the above-mentioned genetically engineered preparation in the synthesis of vanillin.

[0059] In yet another aspect, the present invention provides a method for synthesizing vanillin, the method comprising using the above-mentioned 4-vinylguaiacol oxygenase mutant, the above-mentioned encoding gene, the above-mentioned recombinant vector, the above-mentioned genetically engineered strain or the above-mentioned genetically engineered preparation.

[0060] The method comprises the following steps:

[0061] S1, activating the genetically engineered strain and then culturing the strain to obtain a seed solution;

[0062] S2, inoculating the seed liquid into a fermentation medium for fermentation and culturing;

[0063] 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;

[0064] S4, using 4-vinylguaiacol as a substrate, adding the crude enzyme solution of the genetically engineered strain in step S3 to react, and preparing vanillin.

[0065] The beneficial effects of the present invention are:

[0066] The invention mutates the center site of the pocket of 4-vinylguaiacol oxygenase Iso, and the enzyme activity of the mutant M351F is 1.93 times that before the mutation. At a substrate concentration of 100 mmol / L, the conversion rate of vanillin is 84.72%, and the yield reaches 12.88 g / L. The Iso mutant provided by the invention can efficiently catalyze 4-vinylguaiacol to generate vanillin, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the HPLC detection spectrum of vanillin and 4-vinylguaiacol.

[0068] Figure 2 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.

[0069] Figure 3 The 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 mutant V58W; 3 represents lane 3, which is mutant T216M; 4 represents lane 4, which is mutant T216L; 5 represents lane 5, which is mutant S280M; 6 represents lane 6, which is mutant G307P; 7 represents lane 7, which is mutant M351F. DETAILED DESCRIPTION

[0070] 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.

[0071] The culture medium formula involved in the present invention is as follows:

[0072] 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.

[0073] 2. LB solid medium (1 L): Add 15 g agar to the LB liquid medium formula.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Basic Experimental Example 1 Establishment of Vanillin Detection Method

[0078] Vanillin and 4-vinylguaiacol standard samples were dissolved in 100% methanol solution, and the contents of vanillin and 4-vinylguaiacol were detected by high performance liquid chromatography (HPLC).

[0079] 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.

[0080] The HPLC detection method of vanillin and 4-vinylguaiacol was successfully established. Figure 1 As shown, the elution positions of vanillin and 4-vinylguaiacol are 8.721min and 12.784min respectively, both 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.

[0081] Basic Experimental Example 2 Determination of Enzyme Activity of Vinylguaiacol Oxygenase

[0082] The reaction system for determining enzyme activity was as follows (1 mL): 100 μg of pure enzyme was mixed with 0.5 mol / L pH9.5 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.

[0083] 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.

[0084] Basic Experimental Example 3 Construction of pET28a-Iso, an expression vector of genes related to the vanillin synthesis pathway

[0085] 1. Construction of pET28a-Iso vector

[0086] 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.

[0087] 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 sent to General Biotechnology (Anhui) Co., Ltd. for synthesis, and PCR amplification was performed using primers P1 (Iso-F, Iso-R) to amplify a linear fragment Iso (1482 bp) having a nucleotide sequence as shown in SEQ ID NO. 8.

[0088] SEQ ID NO.8:

[0089]

[0090] The primer sequences used are shown in Table 1:

[0091] Table 1 Primer P1 sequence information

[0092]

[0093] In Table 1, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0094] When amplifying the linearized pET28a vector, the nucleotide sequence of pET28a (as shown in SEQ ID NO. 11) was amplified by PCR using primers P2 (pET28a-F, pET28a-R) to obtain the linearized pET28a vector (5308 bp).

[0095] SEQ ID NO.11:

[0096]

[0097] The primer sequences used are shown in Table 2:

[0098] Table 2 Primer P2 sequence information

[0099] Primers Serial Number sequence pET28a-F SEQ ID NO.12 GCTAGCCATATGGCTGCCG pET28a-R SEQ ID NO.13 GCACTCGAGCACCACCAC

[0100] In Table 2, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0101] The linearized pET28a vector and fragment Iso 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 obtained recombinant plasmid with the correct sequence was recorded as pET28a-Iso. The obtained correct plasmid pET28a-Iso was transformed into E. coli BL21 (DE3) (Weidi Biotechnology, CAT#: EE1002) by the calcium chloride chemical transformation method, and 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).

[0102] 2. Cultivation and induced expression of recombinant strains

[0103] The recombinant strain BL21-pET28a-Iso was inoculated into 2YT medium (containing 50 μg / mL Kana) with a volume of 1 L / 5 L and cultured in a shaker at 37 °C and 220 rpm 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.

[0104] Take 50OD BL21-pET28a-Iso 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-Iso crude enzyme. After the crushing is completed, collect the supernatant, precipitate and crushing liquid for SDS-PAGE electrophoresis to detect protein expression.

[0105] The expression of the recombinant strain BL21-pET28a-Iso enzyme is as follows Figure 2M 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.

[0106] Example 1 Construction of 4-vinylguaiacol oxygenase mutants based on whole plasmid PCR

[0107] Using the recombinant plasmid pET28a-Iso (Basic Experiment Example 3) 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.

[0108] Table 3 Primer sequence information for site-directed mutagenesis

[0109]

[0110]

[0111] In Table 3, F and R are amplification primers, wherein: F represents a forward primer, and R represents a reverse primer.

[0112] After the PCR was completed, 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 Basic Experimental Example 3, spread on LB solid plates (containing 50 μg / mL Kana), and cultured at 37°C overnight. Four transformants were picked and cultured in LB liquid culture medium (containing 50 μg / mL Kana) for 6 h before being sent for sequencing. The correctly sequenced BL21-pET28a-IsoV58W, BL21-pET28a-Iso T216M, BL21-pET28a-IsoT216L, BL21-pET28a-Iso S280M, BL21-pET28a-Iso G307P, and BL21-pET28a-Iso M351F were obtained.

[0113] Example 24 Protein expression and purification of vinylguaiacol oxygenase Iso and its mutants

[0114] 1. Protein expression

[0115] 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 OD600 =0.6-0.8, add IPTG and FeCl2 with a final concentration of 1mmol / L, and induce expression at 25℃ for 12-16h.

[0116] 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.

[0117] 2. Protein Purification

[0118] 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.

[0119] 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 3 As shown, wherein M represents Marker, lane 1 represents the parent Iso of 4-vinylguaiacol oxygenase, lane 2 represents mutant V58W, lane 3 represents mutant T216M, lane 4 represents mutant T216L, lane 5 represents mutant S280M, lane 6 represents mutant G307P, and lane 7 represents mutant M351F.

[0120] Example 3 Comparison of the activity of the Iso mutant and the parent enzyme

[0121] 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 2.80 U / mg, and the specific activities of the mutants V58W, T216M, T216L, S280M, G307P, and M351F were 2.16 U / mg, 6.40 U / mg, 3.07 U / mg, 2.93 U / mg, 3.14 U / mg, and 5.40 U / mg, respectively. The determination results are shown in Table 4.

[0122] Table 4 Comparison of the activities of different Iso mutants and the parent enzyme

[0123] Enzymes Specific activity U / mg Iso 2.80 V58W 2.16 T216M 6.40 T216L 3.07 S280M 2.93 G307P 3.14 M351F 5.40

[0124] Example 4 Comparison of the conversion rate and yield of vanillin synthesized by the Iso mutant and the parent crude enzyme

[0125] 1. Strain culture and crude enzyme solution preparation

[0126] The strain culture and crude enzyme solution preparation refer to "2. Cultivation of recombinant strains and induced expression" in Basic Experimental Example 3.

[0127] 2. Conversion of 4-vinylguaiacol to vanillin

[0128] In a 2L conical flask, add 100mmol / L 4-vinylguaiacol as substrate and 40OD of crude enzyme solution of BL21-pET28a-Iso or its mutants, convert at pH 9.5 and reaction temperature 25°C for 4-5h, and analyze by HPLC.

[0129] The results showed that after 5 hours of reaction, the conversion rate of vanillin of the parent Iso was 63.53% and the yield was 9.66 g / L. The conversion rates of mutants V58W, T216M, T216L, S280M, G307P, and M351F were 38.94%, 88.44%, 68.76%, 67.9%, 70.52%, and 84.72%, respectively; the yields were 5.92 g / L, 13.44 g / L, 10.45 g / L, 10.32 g / L, 10.72 g / L, and 12.88 g / L, respectively. The results are shown in Table 5.

[0130] Table 5 Comparison of conversion rate and yield

[0131] Enzymes Conversion Rate % Yield g / L Iso 63.53％ 9.66 V58W 38.94％ 5.92 T216M 88.44％ 13.44 T216L 68.76％ 10.45 S280M 67.90％ 10.32 G307P 70.52％ 10.72 M351F 84.72％ 12.88

[0132] 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 4-vinylguaiacol oxygenase mutant, characterized in that: The 4-vinylguaiacol oxygenase mutant is a mutant of the pocket center site of 4-vinylguaiacol oxygenase Iso; The amino acid sequence of the 4-vinylguaiacol oxygenase Iso is shown in SEQ ID NO.1, and the center site of the pocket is position 351 of SEQ ID NO.1; The 4-vinylguaiacol oxygenase mutant is: a mutant M351F in which the methionine M at position 351 is mutated to phenylalanine F; the amino acid sequence of the 4-vinylguaiacol oxygenase mutant is shown in SEQ ID NO.

6.

2. A coding gene, characterized in that The coding gene is used to encode the 4-vinylguaiacol oxygenase mutant according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector carries the coding gene according to claim 2.

4. A genetically engineered strain, characterized in that: The genetically engineered strain comprises the recombinant vector according to claim 3, and heterologously overexpresses the Iso mutant.

5. The genetically engineered strain according to claim 4, characterized in that The genetic engineering strain uses the recombinant plasmid pET28a-Iso as a template, uses primers to perform in vitro mutation amplification, and then is transferred into Escherichia coli.

6. A genetic engineering preparation, characterized in that: The genetically engineered preparation comprises one or more of the culture, culture extract, cell disruptor, bacterial body, fermentation broth, fermentation broth precipitate, and freeze-dried powder of the genetically engineered strain according to any one of claims 4 to 5.

7. Use of the 4-vinylguaiacol oxygenase mutant according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3, the genetically engineered strain according to any one of claims 4 to 5, or the genetically engineered preparation according to claim 6 in synthesizing vanillin.

8. A method for synthesizing vanillin, characterized in that: The method comprises using the 4-vinylguaiacol oxygenase mutant according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3, the genetically engineered strain according to any one of claims 4-5 or the genetically engineered preparation according to claim 6.

9. The method according to claim 8, 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 4-vinylguaiacol as a substrate, adding the crude enzyme solution of the genetically engineered strain in step S3 to react, and preparing vanillin.

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