A recombinant strain expressing phenolic acid decarboxylase, its construction method and application

Abstract

The present invention relates to the technical fields of genetic engineering and recombinant enzyme catalysis engineering, and particularly relates to a recombinant strain expressing phenolic acid decarboxylase, a construction method thereof, and applications thereof. The present invention constructs a recombinant strain expressing phenolic acid decarboxylase PAD_Cs by heterologously expressing the encoding gene of phenolic acid decarboxylase PAD_Cs . The enzyme solution of the recombinant strain expressing phenolic acid decarboxylase has remarkable catalytic performance. It can use the enzyme solution induced expression and purified from the recombinant strain to catalyze the decarboxylation reaction of p-coumaric acid with p-coumaric acid as the substrate to synthesize p-hydroxystyrene. At the same time, phenolic acid decarboxylase PAD_Cs has a wide substrate spectrum. It can use 4-vinylphenol compounds as the substrate and bicarbonate as the carboxyl source, and use the freeze-dried whole cells of the recombinant strain to catalyze the regioselective β-carboxylation reaction of 4-vinylphenol compounds, enzymatically synthesize a series of phenolic acids with biological activities while fixing CO2, providing great application potential for the utilization of CO2.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and recombinant enzyme catalytic engineering, and particularly relates to a recombinant strain expressing phenolic acid decarboxylase, a construction method thereof, and applications thereof. Background Art

[0002] Due to the rich reserves, low price, easy availability, and non-toxicity of CO2, it has shown great potential in the fields of green chemistry and organic synthesis, and is regarded as an ideal C1 source for carboxylation and carbonylation reagents. Therefore, the conversion of CO2 into useful chemical substances has attracted extensive attention. So far, researchers have developed various strategies to achieve the conversion and utilization of CO2, including transition metal catalysis, photocatalysis, electrochemical methods, and enzyme catalysis, etc. However, the low energy level of CO2 puts it at a disadvantage in the thermodynamic equilibrium of many chemical processes. In addition, the concentration of CO2 in the atmosphere is low, which does not allow direct chemical reactions, and the actual reaction of CO2 usually needs to exist in the form of bicarbonate in solution, or be carried out under pressurized gas or supercritical state. These adverse factors lead to the disadvantages of traditional chemical engineering strategies such as high cost, low efficiency, high energy consumption, and large environmental impact. In contrast, enzyme catalysis has the characteristics of mild reaction conditions, strong regioselectivity, and strong affinity for substrates, providing significant advantages for the utilization of CO2.

[0003] Carboxylic acids are a crucial class of compounds in pharmaceutical chemistry and the synthesis of fine chemicals. In the process of enzyme catalysis, the direct carboxylation reaction of carbon-hydrogen bonds with carbon dioxide is a more efficient, environmentally friendly, and economical method for producing carboxylic acids. In recent years, some (de)carboxylases have the ability to introduce carboxyl groups in the presence of excessive HCO3 - These (de)carboxylases can be divided into three categories according to their carboxylation mechanisms: divalent metal-dependent (de)carboxylases, coenzyme-independent carboxylases, and prFMN-dependent carboxylases. Among these (de)carboxylases, phenolic acid decarboxylase does not require any cofactors or metal ions for catalysis. At present, there is no other chemical method that can replace phenolic acid decarboxylase to catalyze the β-carboxylation reaction of styrene derivatives to produce coumaric acid, ferulic acid, octanoic acid, etc. Only the method of palladium-catalyzed substitution of 2-hydroxy styrene derivatives has been reported.

[0004] Therefore, it is of great significance to explore phenolic acid decarboxylase with high catalytic activity as a biocatalytic carboxylation tool, construct a recombinant strain with high expression of phenolic acid decarboxylase, and fix CO2. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant strain expressing phenolic acid decarboxylase in view of the deficiencies of the prior art.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned recombinant strain expressing phenolic acid decarboxylase.

[0007] The last technical solution to be solved by the present invention is to provide the application of the above-mentioned recombinant strain.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0009] A recombinant strain expressing phenolic acid decarboxylase is obtained by heterologously expressing the coding gene of phenolic acid decarboxylase PAD_Cs to obtain the recombinant strain.

[0010] Among them, the phenolic acid decarboxylase is derived from Clostridium sp. DSM 8431, and its amino acid sequence is shown in SEQ ID NO: 1.

[0011] Among them, the coding gene of the phenolic acid decarboxylase PAD_Cs has a nucleotide sequence shown in SEQ ID NO: 2.

[0012] The method for constructing the recombinant strain expressing phenolic acid decarboxylase is also within the scope protected by the present invention.

[0013] Specifically, a recombinant plasmid containing the coding gene of phenolic acid decarboxylase PAD_Cs is transformed into a host cell to construct the recombinant strain.

[0014] Among them, the host cell is Escherichia coli.

[0015] In some embodiments of the present invention, the recombinant plasmid containing the coding gene of phenolic acid decarboxylase PAD_Cs uses pET28a(+) as the vector.

[0016] In some embodiments of the present invention, the host cell is Escherichia coli E.coli BL21(DE3).

[0017] The recombinant strain expressing phenolic acid decarboxylase is also within the scope protected by the present invention in the catalytic synthesis of 4-vinylphenol (p-hydroxystyrene).

[0018] Among them, in the catalytic synthesis, 3-(4-hydroxyphenyl)acrylic acid (i.e., p-coumaric acid) is used as the substrate, and the enzyme solution obtained by inducing expression and purification of the recombinant strain is used to catalyze the decarboxylation reaction of 3-(4-hydroxyphenyl)acrylic acid (i.e., p-coumaric acid) to synthesize 4-vinylphenol (p-hydroxystyrene).

[0019] Among them, the 3-(4-hydroxyphenyl)acrylic acid is trans-3-(4-hydroxyphenyl)acrylic acid.

[0020] Among them, the 3-(4-hydroxyphenyl)acrylic acid has the structural formula: .

[0021] Among them, the 4-vinylphenol has the structural formula: .

[0022] Among them, for the catalytic synthesis, the reaction system is: 10~50 mM 3-(4-hydroxyphenyl)acrylic acid (p-coumaric acid), 0.1~1 mg / mL of the enzyme solution after induction expression and purification of the recombinant strain, 50 mM buffer solution with pH 3.0~9.0; for the catalytic synthesis, the conditions are: reacting at 30~70 °C for 5~15 min.

[0023] Specifically, the buffer solution includes: citric acid-sodium citrate buffer solution (pH 3.0~5.5), PB buffer solution (pH 5.5~8.0), Tris-HCl buffer solution (pH 8.0~9.0).

[0024] The application of the recombinant strain expressing phenolic acid decarboxylase in the catalytic synthesis of 3-(4-hydroxyphenyl)acrylic acid compounds is also within the scope protected by the present invention.

[0025] Among them, for the catalytic synthesis, using 4-vinylphenol compounds as substrates and bicarbonate as the carboxyl source, the freeze-dried whole cells of the recombinant strain are used to catalyze the carboxylation reaction of 4-vinylphenol compounds to synthesize 3-(4-hydroxyphenyl)acrylic acid compounds.

[0026] Among them, the 4-vinylphenol compounds have the structural formula: ; the 3-(4-hydroxyphenyl)acrylic acid compounds have the structural formula: .

[0027] Specifically, the R 1 is selected from any one of hydrogen (H), methyl (Me), methoxy (OMe), ethoxy (OE), chlorine (Cl), bromine (Br), and the R 2 is selected from any one of hydrogen (H), methyl (Me), methoxy (OMe).

[0028] Among them, the 3-(4-hydroxyphenyl)acrylic acid compounds are trans-3-(4-hydroxyphenyl)acrylic acid compounds; specifically, "E" in the structural formula of the 3-(4-hydroxyphenyl)acrylic acid compounds represents trans.

[0029] In some embodiments of the present invention, the 4-vinylphenol compounds include any one of 4-vinylphenol (1a, i.e., p-hydroxystyrene), 2-methoxy-4-vinylphenol (2a), 2-ethoxy-4-vinylphenol (3a), 2-methyl-4-vinylphenol (4a), 2-chloro-4-vinylphenol (5a), 2-bromo-4-vinylphenol (6a), 2,6-dimethyl-4-vinylphenol (7a), and 2,6-dimethoxy-4-vinylphenol (8a).

[0030] In some embodiments of the present invention, the corresponding 3-(4-hydroxyphenyl)acrylic acid compounds include any one of trans-3-(4-hydroxyphenyl)acrylic acid (1b, i.e., p-coumaric acid), trans-3-(4-hydroxy-3-methoxyphenyl)acrylic acid (2b, i.e., ferulic acid), trans-3-(4-hydroxy-3-ethoxyphenyl)acrylic acid (3b), trans-3-(4-hydroxy-3-methylphenyl)acrylic acid (4b), trans-3-(4-hydroxy-3-chlorophenyl)acrylic acid (5b), trans-3-(4-hydroxy-3-bromophenyl)acrylic acid (6b), trans-3-(4-hydroxy-3,5-dimethylphenyl)acrylic acid (7b), and trans-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylic acid (8b, i.e., sinapic acid).

[0031] Among them, for the catalytic synthesis, the catalytic reaction is carried out in a CO2 environment, and the reaction system is: 20-40 mg / mL freeze-dried whole cells of the recombinant strain, 100 mM phosphate buffer with a pH of 5.5-7.0, 10-100 mM 4-vinylphenol compounds, 0-20% v / v acetonitrile, and 1-3 M KHCO3; for the catalytic synthesis, the conditions are: reacting at 30-40 °C and 200-1000 rpm for 12-24 h.

[0032] Specifically, the freeze-dried whole cells of the recombinant strain are obtained by centrifuging the fermentation broth induced and expressed by the recombinant strain, collecting the thalli, washing the thalli 2-3 times with pure water, pre-freezing the thalli at -80 °C for 12 h, and then quickly freeze-drying for 12-16 h.

[0033] Beneficial effects: For the new uncharacterized phenolic acid decarboxylase PAD_Cs, the present invention heterologously expresses the coding gene of the phenolic acid decarboxylase PAD_Cs, a recombinant strain expressing phenolic acid decarboxylase PAD_Cs was constructed. This recombinant strain can use 3-(4-hydroxyphenyl)acrylic acid (i.e., p-coumaric acid) as a substrate, and use the enzyme solution induced and purified from the recombinant strain to catalyze the decarboxylation reaction of 3-(4-hydroxyphenyl)acrylic acid (i.e., p-coumaric acid) to synthesize 4-vinylphenol (p-hydroxystyrene); it can also use 4-vinylphenol compounds as substrates and bicarbonate as a carboxyl source, and use the freeze-dried whole cells of the recombinant strain to catalyze the regioselective β-carboxylation reaction of 4-vinylphenol compounds to synthesize 3-(4-hydroxyphenyl)acrylic acid compounds. It solves the problem that most current phenolic acid decarboxylases are only used for decarboxylation reactions, and there are extremely limited phenolic acid decarboxylases that can catalyze the β-carboxylation reaction of 4-vinylphenol compounds to synthesize bioactive phenolic acids. At the same time, by optimizing the optimal temperature and pH of the decarboxylation reaction of phenolic acid decarboxylase PAD_Cs, the phenolic acid decarboxylase PAD_Cs has better catalytic performance. In addition, in the carboxylation reaction of phenolic acid decarboxylase PAD_Cs, the substrate spectrum of the β-carboxylation reaction was expanded, and the yield of the synthesized product p-coumaric acid exceeded 20%, and the yield of ferulic acid exceeded 26%; at a concentration of 80 mM p-hydroxystyrene, the yield of p-coumaric acid could reach up to 9.7 mM at most. It proves that phenolic acid decarboxylase PAD_Cs is a good biocatalytic carboxylation tool and has great potential for fixing CO2. Brief Description of the Drawings

[0034] The following further specifically describes the present invention with reference to the drawings, and the above and / or other advantages of the present invention will become clearer.

[0035] Figure 1 It is the SDS-PAGE electrophoresis analysis of phenolic acid decarboxylase PAD_Cs. Among them, M: Marker; 1: pET-28a (empty vector); 2: crude enzyme solution of pET-28a-PAD_Cs; 3: purified flow-through solution; 4: purified pET-28a-PAD_Cs eluted with 15% Buffer B.

[0036] Figure 2 It is the analysis chart of the optimal temperature for the decarboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs.

[0037] Figure 3 It is the analysis chart of the optimal pH for the decarboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs.

[0038] Figure 4 It is the conversion rate result of the β-carboxylation reaction of phenolic acid decarboxylase PAD_Cs catalyzing different reaction substrates (4-vinylphenol compounds) to synthesize different reaction products under standard β-carboxylation conditions.

[0039] Figure 5It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 1b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0040] Figure 6 It is the mass spectrometry (MS) spectrum of the synthesis of compound 1b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0041] Figure 7 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 2b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0042] Figure 8 It is the mass spectrometry (MS) spectrum of the synthesis of compound 2b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0043] Figure 9 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 3b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0044] Figure 10 It is the mass spectrometry (MS) spectrum of the synthesis of compound 3b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0045] Figure 11 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 5b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0046] Figure 12 It is the mass spectrometry (MS) spectrum of the synthesis of compound 5b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0047] Figure 13 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 6b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0048] Figure 14 It is the mass spectrometry (MS) spectrum of the synthesis of compound 6b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0049] Figure 15 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 7b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0050] Figure 16 It is the mass spectrometry (MS) spectrum of the synthesis of compound 7b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0051] Figure 17 It is the high-performance liquid chromatography (HPLC) chromatogram of the synthesis of compound 8b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0052] Figure 18 It is the mass spectrometry (MS) spectrum of the synthesis of compound 8b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0053] Figure 19 It is the effect of different substrate (1a, namely p-hydroxystyrene) concentrations on the yield of p-coumaric acid synthesized by β-carboxylation of PAD_Cs phenolic acid decarboxylase. Detailed implementation manners

[0054] The following further specifically describes the present invention in combination with the detailed implementation manners, and the above and / or other advantages of the present invention will become clearer.

[0055] In the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0056] Example 1: Construction of a recombinant strain expressing phenolic acid decarboxylase based on Escherichia coli

[0057] To improve the soluble expression of the protein, the codons of the phenolic acid decarboxylase PAD_Cs sequence derived from the Clostridium Clostridium sp. DSM8431 strain were optimized, and the optimized nucleotide sequence is shown in SEQ ID NO:2. Using a forward primer containing Ncol the I restriction enzyme site (5'-TAACTTTAAGAAGGAGATATACCATGGCGATGAAAAACAAAA-3') and a reverse primer containing Xho the I restriction enzyme site (5'-TCAGTGGTGGTGGTGGTGGTGCTCGAGTTTAATTTTTTTATA-3') to amplify the gene of phenolic acid decarboxylase PAD_Cs. The amplified DNA fragment and the vector pET28a(+) were double digested with the restriction enzymes Ncol I and Xho I (purchased from Baoruiyi Biotechnology Co., Ltd.). The digestion reaction was carried out in a 50 μL system and prepared and reacted according to the instructions of the DNA restriction enzyme. After digestion, the DNA fragment was recovered by gel extraction and ligated with the digested vector at a molar ratio of 3:1. The ligation system was prepared according to 10 μL, and the T4 ligase was used to catalyze the ligation reaction, and the reaction was carried out overnight at 16°C to obtain the recombinant plasmid pET28a-PAD_Cs. The above synthesis process was completed by Azenta Life Sciences Company, and the recombinant plasmid pET28a-PAD_Cs was stored in E. coli DH5α.

[0058] According to the relevant instructions of the "yPrp Plasmid DNA Mini Extraction Kit", the plasmid pET-28a-PAD_Cs was extracted. The recombinant plasmid pET28a-PAD_Cs was transformed into Escherichia coli by heat shock method E.coliIn the competent cells of BL21(DE3), it was spread on an LB plate containing 100 μg / mL kanamycin sulfate and cultured at 37 °C for 14 - 16 h. The sequencing results were verified by sequence determination (completed by Anhui General Biology Co., Ltd.) to obtain the recombinant strain E.coli BL21(DE3)-PAD_Cs.

[0059] Example 2: Expression and purification of phenolic acid decarboxylase PAD_Cs

[0060] 1. Induced expression

[0061] The recombinant strain constructed in Example 1 was inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37 °C and 180 rpm to obtain a seed solution. With an inoculation amount of 2% v / v, the seed solution was inoculated into fresh 50 mL of TB liquid medium and cultured at 37 °C and 180 rpm until the OD 600 When it was 0.6 - 1.0, it was taken out, cooled in an ice-water bath for 5 min, and an inducer IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.1 mmol / L was added, and induced expression was carried out at 16 °C and 150 rpm for 20 h to obtain a fermentation broth.

[0062] 2. Purification of phenolic acid decarboxylase PAD_Cs

[0063] Take the fermentation broth of induced expression, centrifuge at 12000 rpm for 20 min, discard the supernatant, then resuspend and wash the cells with 20 mM Tris-HCl (pH 7.5) buffer, centrifuge at 12000 rpm for 20 min, discard the supernatant, resuspend again with the buffer, and then ultrasonically disrupt. The ultrasonically disrupted solution was centrifuged at 12000 rpm for 20 min, and the supernatant (i.e., the crude enzyme solution) was taken for subsequent SDS-PAGE electrophoresis detection.

[0064] Filter the crude enzyme solution through a 0.22 μm filter membrane. Flush the nickel column with Buffer A (20 mM Tris-HCl, pH 7.5) at a flow rate of 2 mL / min until it reaches equilibrium. Inject the protein sample into the injection loop with a syringe, collect the breakthrough peak protein of the sample, and flush the nickel column again with Buffer A (20 mM Tris-HCl, pH 7.5) until no protein is eluted. Use gradient elution method, flush the nickel column with at least 5 times the volume of Buffer B (20 mM Tris-HCl, 500 mM imidazole, pH 7.5) for each gradient, and collect the absorption peak protein of each gradient until no protein is eluted. The protein solution obtained by nickel column purification is used with a pre-packed desalting column from GE to replace the Buffer containing imidazole with 20 mM Tris-HCl (pH 7.5) buffer to remove the imidazole in the protein solution. Verify the protein solutions of different gradients collected by SDS-PAGE. The results show that the target protein eluted with 15% Buffer B has a higher purity. Figure 1 Lane 4 in

[0065] shows the pET-28a-PAD_Cs band eluted with 15% Buffer B. The SDS-PAGE electrophoresis conditions are as follows: the concentration of the stacking gel is 4%, and the concentration of the separating gel is 12.5%; the sample is mixed with the loading buffer in a ratio of 3:1, and loaded onto the gel after a 5-min reaction in a boiling water bath; the electrophoresis instrument is set with an initial voltage of 120 V, and the voltage is increased to 230 V when the sample moves into the separating gel, and the electrophoresis ends when the sample moves to the bottom of the electrophoresis tank.

[0066] The SDS-PAGE electrophoresis analysis results of phenolic acid decarboxylase PAD_Cs are as Figure 1 shown. It can be seen from this that PAD_Cs has an obvious band at 21 kDa, indicating that the molecular weight of PAD_Cs is 21 kDa, which is consistent with the calculated molecular weight, indicating that PAD_Cs is successfully induced and expressed.

[0067] Example 3: Optimization of the decarboxylation reaction of phenolic acid decarboxylase PAD_Cs to produce p-hydroxystyrene

[0068] 1. Optimization of the optimal temperature for the decarboxylation reaction of phenolic acid decarboxylase

[0069] After diluting the purified phenolic acid decarboxylase PAD_Cs enzyme solution to 0.1 mg / mL, in a 1 mL reaction system, add 200 µL of p-coumaric acid solution with a final concentration of 50 mM, 100 µL of 0.1 mg / mL phenolic acid decarboxylase PAD_Cs enzyme solution, and 700 µL of 50 mM citric acid buffer at pH 6.0. After mixing evenly, react at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C for 5 min respectively, then add 2 mL of acetonitrile to terminate the reaction to obtain a mixture. Centrifuge the mixture at 4 °C and 12,000 g for 10 min, and take the supernatant for high performance liquid chromatography (HPLC) analysis to determine the enzyme activity at different reaction temperatures. Taking the highest enzyme activity among the above reaction temperatures as the control, calculate the relative enzyme activity in turn and draw a curve of enzyme activity changing with temperature.

[0070] Among them, the preparation of the p-coumaric acid solution is as follows: Add 8.21 g of accurately weighed p-coumaric acid to a certain amount of pure water, adjust the pH to 7.0 with NaOH solution, stir with a magnetic stirrer and monitor the solution pH with a pH meter during this period. After the p-coumaric acid powder is completely dissolved, make up the volume to 1 L. At this time, the final concentration of the p-coumaric acid solution is 50 mM.

[0071] Definition of enzyme activity unit: The amount of enzyme that produces 1 µmol of p-hydroxystyrene per minute is 1 IU, and the decarboxylase activity unit of phenolic acid decarboxylase is IU / mL.

[0072] The results are as Figure 2 shown. The optimal temperature for the decarboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs is 50 °C. At 50 °C, the enzyme activity reaches the highest value, indicating that the catalytic efficiency of phenolic acid decarboxylase PAD_Cs is the highest at this temperature.

[0073] Taking the phenolic acid decarboxylase CjPAD from Conocephalum japonicum Conocephalum japonicum, the phenolic acid decarboxylase L. brevis PAD from Lactobacillus Lactobacillus brevis RM84, and the phenolic acid decarboxylase BaPAD-Q-426 from Bacillus amyloliquefaciens Bacillus amyloliquefaciens Q-426 as the control. The results show that the optimal temperature of phenolic acid decarboxylase CjPAD is 25 °C, the optimal temperature of phenolic acid decarboxylase L. brevis PAD is 30 °C, and the optimal temperature of phenolic acid decarboxylase BaPAD-Q-426 is 35 °C. Further consulting the literature found that the optimal temperature of phenolic acid decarboxylase is generally 20 - 40 °C, which is much lower than the optimal temperature of phenolic acid decarboxylase PAD_Cs. A higher optimal temperature means that phenolic acid decarboxylase PAD_Cs has better catalytic performance at higher temperatures.

[0074] 2. Determination of the Optimal pH for the Decarboxylation Reaction of Phenolic Acid Decarboxylase

[0075] After diluting the purified phenolic acid decarboxylase PAD_Cs enzyme solution to 0.1 mg / mL, in a 1 mL reaction system, add 200 µL of p-coumaric acid solution with a final concentration of 50 mM, 100 µL of 0.1 mg / mL phenolic acid decarboxylase PAD_Cs enzyme solution, and 700 µL of buffer solution with a pH of 3.0 - 9.0 (50 mM citric acid-sodium citrate buffer with a pH of 3.0 - 5.5, 50 mM PB buffer with a pH of 5.5 - 8.0, 50 mM Tris-HCl buffer with a pH of 8.0 - 9.0). After mixing evenly, react at 50 °C for 5 min, then add 2 mL of acetonitrile to terminate the reaction to obtain a mixture. Centrifuge the mixture at 4 °C and 12,000 g for 10 min, take the supernatant for high-performance liquid chromatography (HPLC) analysis, and determine the enzyme activity under citrate buffer solutions with different pH values. Taking the highest enzyme activity among the above different pH values as a control, calculate the relative enzyme activities in turn, and draw a curve of enzyme activity changing with the pH of the citrate buffer solution. The definition of enzyme activity is the same as above.

[0076] The results are as Figure 3 shown. The optimal pH of phenolic acid decarboxylase PAD_Cs is 5.0. With the increase or decrease of pH, the enzyme activity decreases, and almost loses its activity when pH ≤ 4 and ≥ 9.

[0077] Example 4: Determination and Optimization of the Carboxylation Reaction of Phenolic Acid Decarboxylase PAD_Cs

[0078] 1. Preparation of Freeze-Dried Whole Cells of Recombinant Strains

[0079] Centrifuge the fermentation broth obtained by inducing the expression of recombinant strains at 4 °C and 12,000 rpm for 10 min; discard the centrifuged supernatant, collect the bacterial cells, add an appropriate amount of pure water to resuspend the precipitated bacterial cells, centrifuge again to collect the bacterial cells, and repeat 2 - 3 times; put the precipitated bacterial cells into a -80 °C ultra-low temperature freezer for pre-freezing for 12 h. Quickly put the pre-frozen precipitated bacterial cells into a freeze dryer and freeze-dry for 12 - 16 h to obtain freeze-dried bacterial powder of recombinant strains, that is, freeze-dried whole cells of recombinant strains.

[0080] 2. β-Carboxylation Reactions with Different Substrates

[0081] To investigate whether the phenolic acid decarboxylase PAD_Cs has the ability to catalyze the carboxylation reaction under standard β-carboxylation reaction conditions, different 4-vinylphenol compounds (1a - 8a) substituted with groups having different electronic properties and steric hindrances were used as substrates to determine the conversion rate of the carboxylation reaction. Whether reaction products were formed was determined by comparing with the standard product in high performance liquid chromatography and combined with mass spectrometry. The reaction conversion rate was calculated by plotting the standard curve of the reaction products.

[0082] The specific determination process is as follows: In a 1 mL reaction system, 20 mg / mL of freeze-dried whole cells of the recombinant strain were resuspended in 100 mM phosphate buffer at pH 5.5, shaken and hydrated at 600 rpm and 30 °C for 30 min, and the substrate (1a - 8a) was added directly or from a substrate solution dissolved in acetonitrile, so that the final concentration of the substrate in the reaction system was 10 mM. Then 0 - 20% v / v acetonitrile and 3 M KHCO3 were added, and the reaction was carried out at 30 °C and 600 rpm for 24 h to obtain the reaction solution. The above reaction system was carried out in a sealed glass bottle filled with CO2 (the bottle mouth was wrapped with a sealing film to prevent CO2 escape). An empty host cell without overexpressing phenolic acid decarboxylase was used as a negative control.

[0083] The reaction solution was centrifuged at 13000 rpm for 15 min, and the supernatant was taken. Every 100 μL of the reaction solution was diluted with 1 mL of water / acetonitrile mixture (v / v 1:1), and 3% v / v trifluoroacetic acid (based on the water / acetonitrile mixture, that is, 30 μL was added) was added. After incubation at room temperature for 5 min, it was centrifuged at 13000 rpm for 15 min, and the β-carboxylation reaction conversion was analyzed by high performance liquid chromatography. The reaction products were identified by comparison with the standard product and identified by mass spectrometry.

[0084] Among them, the detection method of the high performance liquid chromatography: All liquid chromatography measurements were carried out using an Agilent HPLC (Model-1200, phase) equipped with C18 (Agilent5 HC-C18, 250×4.6 mm, 5 μm). The column temperature was 24 °C, and the flow rate was 1 mL min -1 , the program ran for 10 min, and the specific mobile phase and UV detection conditions were as follows.

[0085] (1) Compounds 1a, 1b, 2a, 2b, 4a, 4b, 8a, 8b were respectively detected spectrophotometrically at 280 and 320 nm with 0.1% H2O / TFA (0.1%) and 0.1% MeCN / TFA (40∶60, v / v) as the mobile phase.

[0086] (2)Compounds 3a, 3b, 7a, and 7b were respectively detected spectrophotometrically at 280 and 320 nm with 0.1% H2O / TFA (0.1%) and 0.1% (20:80, v / v) as the mobile phase.

[0087] (3)Compounds 5a, 5b, 6a, and 6b were respectively detected spectrophotometrically at 270 and 320 nm with 0.1% H2O / TFA (0.1%) and 0.1% (20:80, v / v) as the mobile phase.

[0088] In the HPLC detection of the β-carboxylation reaction of different substrates, the detection data at a UV wavelength of 320 nm were used to calculate the concentration of the reaction products. Figure 4 Results of the β-carboxylation reaction conversion rate of phenolic acid decarboxylase PAD_Cs catalyzing different reaction substrates (4-vinylphenol compounds) to synthesize different reaction products under standard β-carboxylation conditions; Figure 5 、 7 、9, 11, 13, 15, 17 are the high-performance liquid chromatography (HPLC) chromatograms of phenolic acid decarboxylase PAD_Cs catalyzing the synthesis of compounds 1b - 3b, 5b - 8b; Figure 6 、 8 、10, 12, 14, 16, 18 are the mass spectrometry (MS) spectra of phenolic acid decarboxylase PAD_Cs catalyzing the synthesis of compounds 1b - 3b, 5b - 8b. It can be seen from the figures that the substrates of the carboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs have a very wide range of substitution patterns in the aromatic system. The substitution types at the meta-position of the benzene ring are diverse, including alkyl, alkoxy, and halogen. In the carboxylation reaction, phenolic acid decarboxylase PAD_Cs has the highest conversion rate of 26% for the reaction substrate 2a (2-methoxy-4-vinylphenol), followed by a conversion rate of 20% for the reaction substrate 1a (4-vinylphenol, i.e., p-hydroxypropene); however, the reaction of the reaction substrate 4a (2-methyl-4-vinylphenol) is not obvious, with a conversion rate < 1%.

[0089] Using phenolic acid decarboxylase PAD Lp derived from Lactobacillus plantarum Lactobacillus plantarum and phenolic acid decarboxylase PAD LI derived from Lactococcus lactis Lactoccocus lactis and phenolic acid decarboxylase PAD from Bacillus amyloliquefaciens Bacillus amyloliquefaciensTaking the phenolic acid decarboxylase PAD_Ba as a control, the conversion rates of different substrate spectra were investigated. It was found that the conversion rate of the phenolic acid decarboxylase PAD Lp for synthesizing p-coumaric acid was only 2%; the conversion rate of the phenolic acid decarboxylase PAD LI for synthesizing p-coumaric acid was only 3%, and the conversion rate for synthesizing ferulic acid was only 2%; the conversion rate of the phenolic acid decarboxylase PAD_Ba for synthesizing p-coumaric acid was 18%, indicating that the yield of the phenolic acid decarboxylase PAD_Cs for biologically active phenolic acid compounds such as p-coumaric acid and ferulic acid was at the forefront among similar enzymes. In addition, in the synthesis of p-coumaric acid, the above phenolic acid decarboxylases all required acetonitrile with a volume fraction of more than 20% in the reaction system. When the phenolic acid decarboxylase PAD Cs of the present invention synthesized p-coumaric acid, the specific volume fraction of acetonitrile required was ≤12% v / v, that is, only a lower volume fraction of acetonitrile was needed to obtain a 20% yield of p-coumaric acid. In summary, the phenolic acid decarboxylase PAD_Cs is a good biocatalytic carboxylation tool with great potential for fixing CO2.

[0090] 3. Effects of Different Substrate Concentrations on the Yield of p-Coumaric Acid Synthesized by the β-Carboxylation Reaction of the Phenolic Acid Decarboxylase PAD_Cs

[0091] In a 1 mL reaction system, 20 mg / mL of freeze-dried whole cells of the recombinant strain were resuspended in 100 mM phosphate buffer at pH 5.5, and hydrated by shaking at 600 rpm and 30 °C for 30 min. Compounds 1a (i.e., p-hydroxystyrene) with substrate concentrations of 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM were added respectively, and then co-solvents 0 - 20% v / v acetonitrile and 3 M KHCO3 were added, and the reaction was carried out at 30 °C and 600 rpm for 24 h to obtain the reaction solution. The above reaction system was carried out in a sealed glass bottle with a lid and filled with CO2 (the bottle mouth was wrapped with a sealing film to prevent CO2 escape). The above process was carried out according to the carboxylation reaction measurement process, and the yield of the carboxylation product p-coumaric acid was measured, and a curve of the yield changing with the substrate concentration was plotted.

[0092] The results are as Figure 19 It can be seen that at a p-hydroxystyrene concentration of 80 mM, the yield of p-coumaric acid can reach a maximum of 9.7 mM.

[0093] The present invention provides a recombinant strain expressing phenolic acid decarboxylase and its construction method and application ideas and methods. There are many methods and ways to specifically implement this technical solution. The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. Use of a recombinant strain in the catalytic synthesis of 3-(4-hydroxyphenyl)acrylic acid compounds; Among them, The recombinant strain is obtained by heterologously expressing the coding gene of phenolic acid decarboxylase PAD_Cs ; The coding gene of the phenolic acid decarboxylase PAD_Cs , and its nucleotide sequence is shown in SEQ ID NO:2; Among them, the 3-(4-hydroxyphenyl)acrylic acid compound is trans-3-(4-hydroxyphenyl)acrylic acid or trans-3-(4-hydroxy-3-methoxyphenyl)acrylic acid.

2. The application according to claim 1, characterized in that, For the said catalytic synthesis, using a 4-vinylphenol compound as a substrate and bicarbonate as a carboxyl source, the freeze-dried whole cells of the recombinant strain are used to catalyze the carboxylation reaction of the 4-vinylphenol compound to synthesize 3-(4-hydroxyphenyl)acrylic acid compounds.

3. The application according to claim 2, wherein The 4-vinylphenol compound is 4-vinylphenol or 2-methoxy-4-vinylphenol.

4. The application according to claim 1, characterized in that, For the said catalytic synthesis, the catalytic reaction is carried out in a CO2 environment, and the reaction system is: 20-40 mg / mL of the freeze-dried whole cells of the recombinant strain, 100 mM phosphate buffer with a pH of 5.5-7.0, 10-100 mM of the 4-vinylphenol compound, 0-20% v / v acetonitrile, 1-3 M KHCO3; for the said catalytic synthesis, the conditions are: reacting at 30-40 °C and 200-1000 rpm for 12-24 h.

5. The application according to claim 2 or 4, characterized in that, The freeze-dried whole cells of the recombinant strain are obtained by centrifuging the fermentation broth obtained by inducing the expression of the recombinant strain, collecting the cells, washing the cells 2-3 times with pure water, pre-freezing the cells at -80 °C for 12 h, and then quickly freeze-drying for 12-16 h.

Images