Carboxylase, engineering bacterium and application of carboxylase in production of aromatic carboxylic acid

Abstract

The invention belongs to the field of genetic engineering and biological catalysis, and discloses carboxylase, engineering bacteria and application of the carboxylase in production of aromatic carboxylic acid. The invention discloses a strain of escherichia coli expressing a carboxylase coding gene from marinicaulia flavus, and a construction method of the escherichia coli comprises the following steps: inserting a target protein carboxylase coding gene into a multiple cloning site of a vector pET-28a (+), so as to obtain a recombinant plasmid pET28a-CW354; and introducing the recombinant plasmid into escherichia coli BL21 (DE3) to obtain the engineering bacterium. The invention also protects a method for producing aromatic carboxylic acid, the carboxylase can be used for carboxylating aromatic phenols obtained by depolymerizing lignin, has a wide substrate spectrum, does not need high temperature and high pressure in the whole process, and has the advantages of mild conditions, simplicity in operation, environmental friendliness and the like.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering and biocatalysis, and in particular to a carboxylase, an engineered bacterium and an application thereof in producing aromatic carboxylic acids. The present invention specifically discloses a carboxylase, and discloses its nucleotide sequence, amino acid sequence and application thereof. The carboxylase of the present invention can carboxylate phenols obtained by depolymerizing lignin. Background Art

[0002] At present, it is known that most carboxylases (decarboxylases) use cofactors in the catalytic process, such as thiamine pyrophosphate (TPP), biotin, (enoyl) flavin, pyridoxal and / or divalent metal ions (including Zn 2+ Mg 2+ , Mn 2+ , Fe 2+ and Co 2+ So far, researchers have discovered some carboxylases in microorganisms such as Rhizobium sporomusa MTP-10005, Rhizobium radiobacter WU-0108, Aspergillus oryzae, Fusarium oxysporum, Trichosporon moniliiforme, and Sphingomonas paucimobilis SYK-6, which can catalyze the selective ortho-carboxylation of hydroxyaromatic compounds.

[0003] Plants are the main producers of hydroxyaromatic compounds, as lignin constitutes 20-35% of the mass of lignocellulosic biomass. Due to its unique chemical structure, lignin is considered a renewable source of aromatic compounds. However, currently operating lignocellulosic biorefineries mainly produce pulp, bio-based chemicals, biofuels, electricity and heat, and these processes mainly utilize the hemicellulose / cellulose fraction of wood, while burning lignin as a waste. It is estimated that the annual production of lignin as a by-product of the pulp and paper industry is about 50 million tons. Therefore, research on the development of this carbon resource is gradually gaining attention. In fact, the value-added utilization of lignin fractions is limited by biomass pretreatment and deconstruction strategies, which usually produce materials with complex physical properties. The future development of lignin biorefining will rely on the development of new technological methods that can convert lignin into valuable chemicals.

[0004] Carboxylase can catalyze the synthesis of phthalic acid using phenolic compounds produced by lignin depolymerization as raw materials and carbon dioxide as a carbon source. This process not only produces chemicals with high economic value, but also realizes the effective utilization of lignin and carbon dioxide. The obtained phthalic acid can be further prepared into polyester materials by removing methoxyl groups and phenolic hydroxyl groups and then polymerizing with diamines. This study made full use of the monomers obtained from lignin depolymerization and used carbon dioxide as a carbon source for the reaction, effectively promoting the utilization of carbon dioxide. Therefore, it is crucial to develop a suitable carboxylase. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a carboxylase, an engineered bacterium and its application in the production of aromatic carboxylic acids in view of the deficiencies of the prior art. The present invention clones and expresses a new type of carboxylase from Marinicaulis flavus for the first time, which can carboxylate phenols obtained by depolymerization of lignin and has important application value.

[0006] In order to solve the above technical problems, the present invention discloses a carboxylase, an engineered bacterium and its application in producing aromatic carboxylic acids. The specific technical scheme is as follows:

[0007] An engineered bacterium is an Escherichia coli expressing a carboxylase encoding gene, wherein the carboxylase encoding gene is derived from Marinicaulis flavus, and its nucleotide sequence is shown in SEQ ID No. 1. The present invention obtains a carboxylase gene derived from Marinicaulis flavus by screening from the NCBI database through gene mining, and the amino acid sequence is shown in SEQ ID No. 2.

[0008] In a second aspect, the present invention provides a method for constructing the engineered bacteria described in the first aspect, comprising the following steps: amplifying the carboxylase encoding gene, cloning it into a plasmid vector to obtain a recombinant plasmid, and introducing it into a host bacterium.

[0009] Wherein, the plasmid vector is pET-28a, and the host bacteria is Escherichia coli BL21. Preferably, the construction method is specifically as follows: inserting the target protein carboxylase encoding gene into the multiple cloning site of the vector pET-28a(+) to obtain the recombinant plasmid pET28a-CW354; introducing the recombinant plasmid pET28a-CW354 into Escherichia coli BL21(DE3) to obtain the engineered bacteria. Further preferably, the recombinant plasmid pET28a-CW354 can be specifically a recombinant plasmid obtained by inserting the encoding gene of the target protein between the NdeI and XhoI restriction sites of the vector pET-28a(+).

[0010] In the third aspect, the present invention provides the use of the engineered bacteria described in the first aspect in the preparation of aromatic carboxylic acids. Preferably, the aromatic carboxylic acid includes any one of 5-carboxyvanillic acid, 5-(2-carboxyvinyl)-2-hydroxybenzoic acid, 2-hydroxy-3-methoxy-5-propylbenzoic acid, 2-hydroxy-3-methoxy-5-methylbenzoic acid and 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid. Further preferably, the present invention provides the use of the engineered bacteria described in the first aspect in the preparation of 5-carboxyvanillic acid using vanillic acid as a substrate.

[0011] Wherein, the engineering bacteria for preparing aromatic carboxylic acid comprises the following steps:

[0012] (1) preparing pure carboxylase enzyme solution: purifying the wet bacterial cells obtained by inducing expression of the engineered bacteria to obtain pure carboxylase enzyme;

[0013] (2) The pure carboxylase prepared in step (1) is placed in a solvent for reaction with a phenolic reaction substrate, a metal ion salt and a bicarbonate to obtain an aromatic carboxylic acid product.

[0014] Wherein, in step (1), the specific method of inducing expression is as follows: inoculate the engineered bacteria into the culture medium, and culture at 30-37°C until OD 600 When the OD value is 0.6 to 0.8, add IPTG with a final concentration of 0.1 to 1 mmol / L, induce expression at 16 to 25°C for 12 to 24 hours, and collect wet cells by centrifugation for purification. Preferably, the engineered bacteria are inoculated into the culture medium and cultured at 37°C until the OD value is 600 When the concentration of IPTG is 0.6 to 0.8, add IPTG at a final concentration of 0.1 mmol / L, induce expression at 25°C for 12 hours, and collect wet cells by centrifugation for purification. Further preferably, a single colony of the engineered bacteria is picked and inoculated into 20 mL of LB liquid culture medium containing 50 μg / mL kanamycin (Kana), shake-cultured at 37°C and 200 rpm for 12 hours to obtain a seed solution, inoculated into 400 mL of LB liquid culture medium containing 50 μg / mL kanamycin at an inoculum volume of 5% v / v, and shake-cultured at 37°C and 200 rpm until OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mmol / L at 25°C for induction for 12 hours, and collect by centrifugation to obtain wet cells. More preferably, the purification comprises washing and crushing the wet cells and then purifying them using a protein purification column and gradient elution method.

[0015] Wherein, in step (2), the phenolic reaction substrate includes any one of vanillic acid, 3-(4-hydroxyphenyl) acrylic acid, 2-methoxy-4-propylphenol, 2-methoxy-4-methylphenol, and 3-(3-methoxy-4-hydroxyphenyl) acrylic acid, which correspond to the following aromatic carboxylic acid products in order: 5-carboxyvanillic acid, 5-(2-carboxyvinyl)-2-hydroxybenzoic acid, 2-hydroxy-3-methoxy-5-propylbenzoic acid, 2-hydroxy-3-methoxy-5-methylbenzoic acid, and 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid;

[0016] The metal ion salt is Zn 2+ Mg 2+ , Mn 2+ , Fe 2+ and Co 2+ Any salt; preferably Mn 2+ The bicarbonate is potassium bicarbonate or sodium bicarbonate; preferably potassium bicarbonate.

[0017] The solvent is a potassium phosphate buffer with a pH of 5.8 to 7.0, preferably, the solvent is a 50 mM potassium phosphate (KPi) buffer with a pH of 5.8.

[0018] Wherein, in the reaction system of step (2), the concentration of pure carboxylase is 0.5-6.0 mg / mL, the concentration of phenolic reaction substrate is 5-20 mM, the concentration of metal ion salt is 0.5-1 mM, and the concentration of bicarbonate is 2.7-3 M. Preferably, the concentration of pure carboxylase is 0.6 mg / mL, the concentration of phenolic reaction substrate is 10 mM, the concentration of metal ion salt is 1 mM, and the concentration of bicarbonate is 2.7 M.

[0019] In step (2), the reaction temperature is 25-37°C and the reaction time is 12-24h. Preferably, the reaction is carried out at 30°C and 200rpm for 24h.

[0020] Beneficial effects:

[0021] The invention screens 5-carboxyvanillic acid decarboxylase from different sources, clones a carboxylase gene from Marinicaulis flavus, uses Escherichia coli engineering bacteria for heterologous expression, and conducts application research.

[0022] The method provided by the present invention is used to prepare aromatic carboxylic acid compounds including but not limited to 5-carboxyvanillic acid. The carboxylase has a wide substrate spectrum, and the whole catalytic process does not require the addition of an inducer, and the whole process does not require high temperature and high pressure. It has the advantages of mild conditions, simple operation, and environmental friendliness. With vanillic acid as a substrate, the pure carboxylase of the present invention can catalyze vanillic acid to prepare 5-carboxyvanillic acid with a yield of 16.9%; with 3-(3-methoxy-4-hydroxyphenyl) acrylic acid as a substrate, the pure carboxylase of the present invention can catalyze 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid with a yield of 19.4%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0024] Figure 1 This is the SDS-PAGE detection result of carboxylase CW354. DETAILED DESCRIPTION

[0025] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0026] In the following examples, the analysis methods of the substrate and aromatic carboxylic acid products are as follows:

[0027] HPLC detection method: The treated solution was analyzed using an Agilent series high performance liquid chromatograph, an Agilent Eclipse Plus C18 column, phase A: chromatographic grade acetonitrile, phase B: 0.3% v / v trifluoroacetic acid aqueous solution, mobile phase ratio: acetonitrile: 0.3% v / v trifluoroacetic acid aqueous solution (v:v) = 2:8, column temperature 40°C, flow rate 0.4 mL / min, wavelength 254 nm.

[0028] The reaction system in the following examples is as follows:

[0029] The reaction adopts 1mL reaction system, 690μL potassium phosphate buffer (KPi buffer, pH=5.8, 50mM) adds 200μL pure enzyme solution (addition concentration 0.6mg / mL), 10μL manganese chloride aqueous solution (addition concentration 1mM), 100μL substrate solution (addition concentration is 10mM) is added, and then 270mg KHCO3 (addition concentration 2.7M, final pH value of reaction system 8.5) is added. In order to avoid the loss of carbon dioxide, the vial is tightly closed with a diaphragm immediately, and the mixture is shaken at 30°C and 200rpm for 24 hours. Thereafter, 100μL reaction mixture of aliquots is added to 900μL water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid volume ratio = 1:1:0.1) to quench the reaction. The sample is incubated at room temperature for 5 minutes and centrifuged (13000rpm, 15 minutes).

[0030] Example 1 Construction and induced expression of carboxylase genetic engineering bacteria

[0031] The gene encoding the carboxylase CW354 from Marinicaulis flavus was synthesized by General Biosystems (Anhui) Co., Ltd. The nucleotide sequence of the gene encoding the carboxylase CW354 is shown in SEQ ID No. 1. The amino acid sequence of the carboxylase CW354 is shown in SEQ ID No. 2:

[0032] The synthesized carboxylase CW354 encoding gene was cloned between the NdeI and XhoI restriction sites on the pET-28a(+) vector to obtain the recombinant plasmid pET28a-CW354, and then the recombinant plasmid was transferred into the competent cells of Escherichia coli BL21(DE3), and then smeared on the LB solid medium plate containing 50μg / mL Kana resistance after shaking culture at 37°C for 1h. After culturing at 37°C for 16h, the single clone on the plate was picked, and it was verified that the carboxylase gene engineering bacteria were constructed.

[0033] Select the correct single colony on the plate and inoculate it into 20mL LB medium containing 50μg / mL Kana resistance. Culture it for about 12h as seed solution. Inoculate it into 400mL LB medium containing 50μg / mL Kana resistance at a 5% v / v inoculum. Culture it on a shaker at 37℃ and 200rpm until OD 600= 0.6 to 0.8, add IPTG with a final concentration of 0.1 mmol / L and induce at 25 ° C for 12 h. After the induction, the bacterial solution was centrifuged at 4 ° C and 8000 rpm for 5 min to collect the wet bacteria, and the bacteria were cleaned using 50mM, pH = 7 KPi buffer (0.8L of distilled water was loaded into a suitable container, 2.829g of potassium dihydrogen phosphate was added to the solution, and 5.087g of potassium dihydrogen phosphate was added. After complete dissolution, distilled water was added until the volume was 1L) and stored at 4 ° C. The bacterial mud was resuspended in 20mL of 50mM pH = 7.4 KPi buffer, and the resuspended bacteria were crushed with an ultrasonic crusher on ice (on 3s, off 5s, power 300W), and crushed for 15 minutes. Then centrifuged (9000rpm, 4 ° C, 40min), the supernatant was collected, and the supernatant was filtered using a 0.22μm water filter to obtain a crude carboxylase enzyme solution.

[0034] 2mL Ni-NTA agarose purification resin filler was loaded into a gravity protein purification column (purchased from Yisheng Bio), and the filler was balanced three times with KPi buffer (50mM pH = 7.4), and then an appropriate amount of crude enzyme solution was slowly added, and gently shaken for about 5 minutes to promote the full adsorption of the enzyme and the filler. Use 20-500mM imidazole buffer (17.01g imidazole was weighed and dissolved in 500mL ultrapure water, 500mM imidazole buffer was obtained by dissolving, and other concentrations of imidazole buffer were obtained by dilution) for gradient elution. The specific process of gradient elution is as follows: the recombinant protein is recovered by the gradient method, and the proportion of elution buffer is gradually increased until the recombinant protein is eluted. Specifically, 20mM imidazole buffer was added to rinse twice, and then 50mM imidazole buffer was used to rinse once to remove impurities; then the target protein was eluted with 200mM imidazole buffer, and the eluate was collected with a centrifuge tube; finally, 500mM imidazole buffer was used to remove the remaining protein in the purification column filler. The collected eluate was concentrated and desalted using a 30kDa MWCO Millipore ultrafiltration tube to obtain a pure enzyme solution. The enzyme activity was detected to be 5.7U / mg. 200μL per tube was dispensed and stored in an ultra-low temperature refrigerator at -80℃. The purity and size were preliminarily verified by polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 As shown, the molecular weight of carboxylase CW354 is 38.1 KDa.

[0035] Example 2 Synthesis of 5-carboxyvanillic acid catalyzed by vanillinase

[0036] The present embodiment is carried out under the carboxylase optimal reaction condition, and the structural formula of reaction substrate and corresponding product is as shown in table 1.Reaction adopts 1mL reaction system, in 690μL KPi (pH=5.8,50mM) buffer, add 200μL pure enzyme liquid (addition concentration 0.6mg / mL) respectively, 10μL manganese chloride aqueous solution (addition concentration 1mM), the vanillic acid (3-methoxy-4-hydroxybenzoic acid, addition concentration 10mM) of 100μL, then add 270mg KHCO3 (addition concentration 2.7M, final pH value 8.5 of reaction system).In order to avoid the loss of carbon dioxide, use diaphragm to close bottle tightly immediately, and mixture is shaken in 30 ℃ and 200rpm shaking table 24 hours.After this, the 100μL reaction mixture of aliquot is added to the water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid volume ratio=1:1:0.1) of 900μL to react with cancellation. The sample was incubated at room temperature for 5 minutes and centrifuged (8000 rpm, 5 minutes). The supernatant was passed through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution, and then HPLC was performed to detect the yield of the carboxylic acid product (5-carboxyvanillic acid).

[0037] The product characterization results are as follows: 1 H NMR (400 MHz, DMSO-D6) δ12.6(br,s,1H),8.01(d,J=1.9Hz,1H),7.60(d,J=1.9Hz,1H),3.85(s,3H); 13 C NMR (100MHz, DMSO-D6) δ171.8,166.5,155.4,148.1,123.5,120.9,116.1,112.9,55.9.

[0038] The yield of the product 5-carboxyvanillic acid was determined to be 16.9%.

[0039] Example 3 Synthesis of 5-(2-carboxyvinyl)-2-hydroxybenzoic acid catalyzed by 3-(4-hydroxyphenyl)acrylase

[0040] The present embodiment is carried out under the carboxylase optimal reaction condition, and the structural formula of reaction substrate and corresponding product is as shown in table 1.Reaction adopts 1mL reaction system, in 690 μ L KPi (pH=5.8,50mM) buffer, add 200 μ L pure enzyme liquid (addition concentration 0.6mg / mL) respectively, 10 μ L manganese chloride aqueous solution (addition concentration 1mM), 100 μ L 3-(4-hydroxyphenyl) acrylic acid (addition concentration 10mM), then add 270mg KHCO (addition concentration 2.7M, reaction system pH=8.5).In order to avoid the loss of carbonic acid gas, use diaphragm to tightly close bottle immediately, and mixture was shaken 24 hours at 30 ℃ and 200rpm shaking table.After this, 100 μ L reaction mixture of aliquot is added water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid=1:1:0.1) of 900 μ L to react with cancellation. The sample was incubated at room temperature for 5 minutes and centrifuged (8000 rpm, 5 minutes). The supernatant was passed through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution, and then HPLC was performed to detect the yield of the carboxylic acid product 5-(2-carboxyvinyl)-2-hydroxybenzoic acid.

[0041] The product characterization results are as follows: 1 H NMR (400MHz, DMSO-D6) δ8.02(d,J=2.0Hz,1H),7.89(dd,J=8.6,2.0Hz,1H),7.56(d,J=16.0Hz,1H),6.99(d,J=8.6Hz,1H),6.39(d,J=16.0Hz,1H); 13 C NMR (100MHz, DMSO-D6) δ171.5,167.7,162.6,143.0,134.5,131.1,125.6,118.0,117.4,113.6.

[0042] It was determined that the yield of the product 5-(2-carboxyvinyl)-2-hydroxybenzoic acid was 13.2%.

[0043] Example 4 Enzymatic Synthesis of 2-Hydroxy-3-methoxy-5-propylbenzoic Acid by 2-Methoxy-4-propylphenol

[0044] The present embodiment is carried out under the carboxylase optimal reaction condition, and the structural formula of reaction substrate and corresponding product is as shown in table 1.Reaction adopts 1mL reaction system, in 690 μ L KPi (pH=5.8,50mM) buffer, add 200 μ L pure enzyme liquid (addition concentration 0.6mg / mL) respectively, 10 μ L manganese chloride aqueous solution (addition concentration 1mM), 100 μ L 2-methoxy-4-propylphenol (addition concentration 10mM), then add 270mg KHCO (addition concentration 2.7M, reaction system pH value 8.5).In order to avoid the loss of carbonic acid gas, use diaphragm to tightly close bottle immediately, and mixture was shaken 24 hours at 30 ℃ and 200rpm shaking table.After this, 100 μ L reaction mixtures of aliquots are added to the water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid=1:1:0.1) of 900 μ L to react with cancellation. The sample was incubated at room temperature for 5 minutes and centrifuged (8000 rpm, 5 minutes). The supernatant was passed through a 0.22 μm organic membrane to remove proteins and other solid impurities in the reaction solution, and then HPLC was performed to detect the yield of the carboxylic acid product (2-hydroxy-3-methoxy-5-propylbenzoic acid).

[0045] The product characterization results are as follows: 1 H NMR (400MHz, CD3OD) δ7.27(d,J=2.0Hz,1H),7.00(d,J=2.0Hz,1H),3.85(s,3H),2.54(t,J=7.4Hz,2H),1.68–1.59(m,2H),0.94(t,J=7.4Hz,3H); 13 C NMR (100MHz, CD3OD) δ176.1,151.3,149.4,134.1,122.0,118.7,114.1,55.7,38.5,25.7,14.0.

[0046] It was determined that the yield of the product 2-hydroxy-3-methoxy-5-propylbenzoic acid was 3.6%.

[0047] Example 5 Enzymatic Synthesis of 2-Hydroxy-3-methoxy-5-methylbenzoic Acid by 2-Methoxy-4-Methylphenol

[0048] The present embodiment is carried out under the carboxylase optimal reaction condition, and the structural formula of reaction substrate and corresponding product is as shown in table 1.Reaction adopts 1mL reaction system, in 690 μ L KPI (pH=5.8,50mM) buffer, add 200 μ L pure enzyme liquid (final concentration 0.6mg / mL) respectively, 10 μ L manganese chloride solution (final concentration 1mM), 100 μ L 2-methoxy-4-methylphenol (final concentration 10mM), then add 270mg KHCO (addition concentration 2.7M, final pH value 8.5 of reaction system).In order to avoid the loss of carbonic acid gas, use diaphragm to tightly close bottle immediately, and mixture is shaken 24 hours at 30 ℃ and 200rpm shaking table.After this, 100 μ L reaction mixture of aliquot is added water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid=1:1:0.1) of 900 μ L to react with cancellation. The sample was incubated at room temperature for 5 minutes and centrifuged (8000 rpm, 5 minutes). The supernatant was passed through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution and then subjected to HPLC detection to detect the yield of the carboxylic acid product.

[0049] The product characterization results are as follows: 1 H NMR (400MHz, CDCl3) δ10.36(s,1H),7.31(d,J=1.6Hz,1H),6.92(d,J=1.6Hz,1H),3.91(s,3H),2.32(s,3H); 13 C NMR (100MHz, CDCl3) δ174.6,150.5,148.4,128.6,121.5,119.1,111.2,56.4,21.1.

[0050] It was determined that the yield of the product 2-hydroxy-3-methoxy-5-methylbenzoic acid was 7.3%.

[0051] Example 6 Synthesis of 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid catalyzed by 3-(3-methoxy-4-hydroxyphenyl)acrylase

[0052] The present embodiment is carried out under the carboxylase optimal reaction condition, and the structural formula of reaction substrate and corresponding product is as shown in table 1.Reaction adopts 1mL reaction system, in 690μL KPi (pH=5.8,50mM) buffer, add 200μL pure enzyme liquid (addition concentration 0.6mg / mL) respectively, 10μL manganese chloride aqueous solution (addition concentration 1mM), 100μL 3-(3-methoxyl group-4-hydroxyphenyl) acrylic acid (addition concentration 10mM), then add 270mg KHCO3 (addition concentration 2.7M, final pH value 8.5 of reaction system).In order to avoid the loss of carbonic acid gas, use diaphragm to close bottle tightly immediately, and mixture is shaken 24 hours at 30 ℃ and 200rpm shaking table.After this, 100μL reaction mixture of aliquot is added to the water-acetonitrile-trifluoroacetic acid mixture (water: acetonitrile: trifluoroacetic acid volume ratio=1:1:0.1) of 900μL to react with cancellation. The sample was incubated at room temperature for 5 minutes and centrifuged (8000 rpm, 5 minutes). The supernatant was passed through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution and then subjected to HPLC detection to detect the yield of the carboxylic acid product.

[0053] The product characterization results are as follows: 1 H NMR (400MHz, DMSO-D6) δ7.58(d,J=1.6Hz,1H),7.55(d,J=1.6Hz,1H),7.52(d,J=15.9Hz,1H),6.48(d,J=15.9Hz,1H),3.86(s,3H); 13 C NMR (100MHz, DMSO-D6) δ172.0,167.8,153.6,148.7,143.5,125.1,123.0,117.6,114.5,113.1,56.1.

[0054] It was determined that the yield of the product 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid was 19.4%.

[0055] Table 1 Substrates and corresponding product structures described in Examples 2 to 6

[0056]

[0057] The present invention provides a carboxylase, an engineering bacterium and a method for using the same in producing aromatic carboxylic acids. There are many methods and approaches for realizing the technical solution. The above is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. An engineered bacterium, characterized in that: The engineered bacteria is Escherichia coli expressing a gene encoding carboxylase; The carboxylase encoding gene is derived from Marinicaulis flavus, and its nucleotide sequence is shown in SEQ ID No.

1.

2. The method for constructing the engineering bacteria according to claim 1, characterized in that: The method comprises the following steps: amplifying the carboxylase encoding gene, cloning it into a plasmid vector to obtain a recombinant plasmid, and introducing it into a host bacterium to obtain the obtained plasmid.

3. The construction method according to claim 2, characterized in that: The plasmid vector is pET-28a, and the host bacteria is Escherichia coli BL21.

4. Use of the engineered bacteria according to claim 1 in the preparation of aromatic carboxylic acids.

5. The use according to claim 4, characterized in that: The aromatic carboxylic acid includes any one of 5-carboxyvanillic acid, 5-(2-carboxyvinyl)-2-hydroxybenzoic acid, 2-hydroxy-3-methoxy-5-propylbenzoic acid, 2-hydroxy-3-methoxy-5-methylbenzoic acid and 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid.

6. The use according to claim 4, characterized in that: The engineering bacteria for preparing aromatic carboxylic acids comprises the following steps: (1) Preparing pure carboxylase enzyme: purifying the wet bacterial cells obtained by inducing expression of the engineered bacteria to obtain pure carboxylase enzyme; (2) The pure carboxylase prepared in step (1) is placed in a solvent for reaction with a phenolic reaction substrate, a metal ion salt and a bicarbonate to obtain an aromatic carboxylic acid product.

7. The use according to claim 6, characterized in that: In step (1), the induction expression is specifically carried out as follows: the engineered bacteria are inoculated into a culture medium and cultured at 30-37°C until the OD 600 When the pH value is 0.6-0.8, add IPTG with a final concentration of 0.1-1 mmol / L, induce expression at 16-25°C for 12-24 hours, and collect the wet cells by centrifugation for purification.

8. The use according to claim 6, characterized in that: In step (2), the phenolic reaction substrate includes any one of vanillic acid, 3-(4-hydroxyphenyl) acrylic acid, 2-methoxy-4-propylphenol, 2-methoxy-4-methylphenol, and 3-(3-methoxy-4-hydroxyphenyl) acrylic acid, which correspond to the following aromatic carboxylic acid products in order: 5-carboxyvanillic acid, 5-(2-carboxyvinyl)-2-hydroxybenzoic acid, 2-hydroxy-3-methoxy-5-propylbenzoic acid, 2-hydroxy-3-methoxy-5-methylbenzoic acid, and 5-(2-carboxyvinyl)-2-hydroxy-3-methoxybenzoic acid; The metal ion salt is Zn 2+ Mg 2+ , Mn 2+ , Fe 2+ and Co 2+ The bicarbonate is potassium bicarbonate or sodium bicarbonate; the solvent is potassium phosphate buffer with a pH of 5.8 to 7.

0.

9. The use according to claim 6, characterized in that: In the reaction system of step (2), the concentration of pure carboxylase is 0.5-6.0 mg / mL, the concentration of phenol reaction substrate is 5-20 mM, the concentration of metal ion salt is 0.5-1 mM, and the concentration of bicarbonate is 2.7-3 M.

10. The use according to claim 6, characterized in that: In step (2), the reaction temperature is 25 to 37° C. and the reaction time is 12 to 24 hours.

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