Recombinant strain for expressing phenolic acid decarboxylase as well as construction method and application of recombinant strain

Abstract

The invention relates to the technical field of gene engineering and recombinase catalysis engineering, in particular to a recombinant strain for expressing phenolic acid decarboxylase as well as a construction method and application of the recombinant strain. According to the invention, a recombinant strain for expressing the phenolic acid decarboxylase PADCs is constructed by heterologous expression of a coding gene PADCs of the phenolic acid decarboxylase. The enzyme liquid of the recombinant strain for expressing the phenolic acid decarboxylase has remarkable catalytic performance, p-coumaric acid can be used as a substrate, the recombinant strain is used for inducing expression, the purified enzyme liquid is used for catalyzing decarboxylation reaction of p-coumaric acid, and p-hydroxystyrene is synthesized; meanwhile, the phenolic acid decarboxylase PADCs has a wide substrate spectrum, the 4-vinylphenol compound can be used as a substrate, bicarbonate is used as a carboxyl source, the freeze-dried whole cell of the recombinant strain is utilized to catalyze the 4-vinylphenol compound to carry out regioselective beta-carboxylation reaction, a series of phenolic acids with biological activity are synthesized by an enzymic method, and meanwhile CO2 is immobilized; and a huge application potential is provided for the utilization of CO2.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering and recombinase catalytic engineering, and in particular to a recombinant strain expressing phenolic acid decarboxylase and a construction method and application thereof. Background Art

[0002] Due to its abundant reserves, low price, easy availability and non-toxicity, CO2 has shown great potential in the field of green chemistry and organic synthesis and is regarded as an ideal C1 source carboxylation and carbonylation reagent. Therefore, the conversion of CO2 into useful chemicals has attracted widespread attention. To date, researchers have developed a variety of strategies to achieve the conversion and utilization of CO2, including transition metal catalysis, photocatalysis, electrochemical methods and enzyme catalysis. However, the low energy level of CO2 puts it at a disadvantage in the thermodynamic equilibrium of many chemical processes. In addition, the low concentration of CO2 in the atmosphere 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 in a pressurized gas or supercritical state. These unfavorable factors lead to the disadvantages of traditional chemical 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, which provides significant advantages for the utilization of CO2.

[0003] Carboxylic acids are a class of compounds that are of vital importance in the synthesis of medicinal chemistry and fine chemicals. In an enzyme-catalyzed process, carbon-hydrogen bonds are directly carboxylated with carbon dioxide, which is a more efficient, environmentally friendly and economical method for producing carboxylic acids. In recent years, some (de)decarboxylases have been developed in the presence of excess HCO3 - It has the ability to introduce carboxyl groups in the presence of phenol. These (de)carboxylases can be divided into three categories based on their carboxylation mechanisms: divalent metal-dependent (de)carboxylases, coenzyme-independent carboxylases, and prFMN-dependent carboxylases. Among these (de)carboxylases, phenolic acid decarboxylases do not require any auxiliary factors or metal ions for catalysis. Currently, 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 palladium-catalyzed substitution of 2-hydroxystyrene 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, and to construct a recombinant strain that efficiently expresses phenolic acid decarboxylase and fixes 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] The technical problem that the present invention also solves is to provide a method for constructing the above-mentioned recombinant strain expressing phenolic acid decarboxylase.

[0007] The final 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 solution adopted by the present invention is as follows: A recombinant strain expressing phenolic acid decarboxylase, wherein the coding gene of phenolic acid decarboxylase is heterologously expressed PAD_Cs , and obtain the recombinant strain.

[0009] Wherein, the phenolic acid decarboxylase is derived from Clostridium sp. DSM 8431, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] Wherein, the coding gene of the phenolic acid decarboxylase PAD_Cs , whose nucleotide sequence is shown in SEQ ID NO:2.

[0011] The method for constructing the recombinant strain expressing phenolic acid decarboxylase is also within the scope of protection of the present invention.

[0012] Specifically, the gene encoding phenolic acid decarboxylase PAD_Cs The recombinant plasmid is transformed into a host cell to construct the recombinant strain.

[0013] Wherein, the host cell is Escherichia coli.

[0014] In some embodiments of the present invention, the gene encoding phenolic acid decarboxylase PAD_Cs The recombinant plasmid was constructed with pET28a(+) as the vector.

[0015] In some embodiments of the present invention, the host cell is Escherichia coli E. coli BL21(DE3).

[0016] The recombinant strain expressing phenolic acid decarboxylase catalyzes the synthesis of 4-vinylphenol (p-hydroxystyrene) and is also within the scope of protection of the present invention.

[0017] The catalytic synthesis uses 3-(4-hydroxyphenyl) acrylic acid (i.e., p-coumaric acid) as a substrate, and utilizes the recombinant strain to induce expression and the purified enzyme solution to catalyze the decarboxylation reaction of 3-(4-hydroxyphenyl) acrylic acid (i.e., p-coumaric acid) to synthesize 4-vinylphenol (p-hydroxystyrene).

[0018] Wherein, the 3-(4-hydroxyphenyl)acrylic acid is trans-3-(4-hydroxyphenyl)acrylic acid.

[0019] Wherein, the 3-(4-hydroxyphenyl) acrylic acid has the structural formula: .

[0020] Wherein, the structural formula of the 4-vinylphenol is: .

[0021] The reaction system of the catalytic synthesis is: 10-50 mM 3-(4-hydroxyphenyl)acrylic acid (p-coumaric acid), 0.1-1 mg / mL recombinant strain induced expression, purified enzyme solution, 50 mM pH 3.0-9.0 buffer; the conditions of the catalytic synthesis are: reaction at 30-70°C for 5-15 min.

[0022] Specifically, the buffer includes: citric acid-sodium citrate buffer (pH 3.0-5.5), PB buffer (pH 5.5-8.0), and Tris-HCl buffer (pH 8.0-9.0).

[0023] The use of the recombinant strain expressing phenolic acid decarboxylase in catalyzing the synthesis of 3-(4-hydroxyphenyl) acrylic acid compounds is also within the scope of protection of the present invention.

[0024] The catalytic synthesis uses 4-vinylphenol compounds as substrates and bicarbonate as a carboxyl source, and utilizes freeze-dried whole cells of the recombinant strain to catalyze the carboxylation reaction of 4-vinylphenol compounds to synthesize 3-(4-hydroxyphenyl) acrylic acid compounds.

[0025] Wherein, the 4-vinylphenol compound has the structural formula: ; The 3-(4-hydroxyphenyl) acrylic acid compound has the structural formula: .

[0026] Specifically, the R 1 is selected from any one of hydrogen (H), methyl (Me), methoxy (OMe), ethoxy (OE), chlorine (Cl), and bromine (Br), wherein R 2 Any one selected from hydrogen (H), methyl (Me), and methoxy (OMe).

[0027] Wherein, the 3-(4-hydroxyphenyl) acrylic acid compound is a trans-3-(4-hydroxyphenyl) acrylic acid compound; specifically, in the structural formula of the 3-(4-hydroxyphenyl) acrylic acid compound, "E" represents trans.

[0028] In some embodiments of the present invention, the 4-vinylphenol compound includes 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).

[0029] 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., sinapinic acid).

[0030] Wherein, 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 pH 5.5~7.0 phosphate buffer, 10~100 mM 4-vinylphenol compound, 0~20% v / v acetonitrile, 1~3 M KHCO3; the conditions of the catalytic synthesis are: 30~40°C, 200~1000 rpm, reaction time 12~24 h.

[0031] Specifically, the freeze-dried whole cells of the recombinant strain are obtained by centrifuging the fermentation broth obtained by inducing expression of the recombinant strain, collecting the cells, washing them with pure water 2 to 3 times, pre-freezing the cells at -80°C for 12 hours, and then rapidly freeze-drying them for 12 to 16 hours.

[0032] Beneficial effect: For the new phenolic acid decarboxylase PAD_Cs that has not yet been characterized, the present invention heterologously expresses the coding gene of phenolic acid decarboxylase PAD_CsA recombinant strain expressing phenolic acid decarboxylase PAD_Cs was constructed. The 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 by 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. This solves the problem that most of the current phenolic acid decarboxylases are only used for decarboxylation reactions, while there are extremely limited phenolic acid decarboxylases that can catalyze the β-carboxylation reaction of 4-vinylphenol compounds to synthesize biologically active 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 β-carboxylation reaction was expanded, and the yield of the synthetic 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 a maximum of 9.7 mM. It was proved that phenolic acid decarboxylase PAD_Cs is a good biocatalytic carboxylation tool with great potential for fixing CO2. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0034] Figure 1 This is the SDS-PAGE electrophoresis analysis of phenolic acid decarboxylase PAD_Cs. Among them, M: Marker; 1: pET-28a (empty vector); 2: pET-28a-PAD_Cs crude enzyme solution; 3: purified flow-through solution; 4: purified pET-28a-PAD_Cs eluted with 15% Buffer B.

[0035] Figure 2 This is the optimal temperature analysis diagram for the decarboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs.

[0036] Figure 3 This is the optimal pH analysis diagram for the decarboxylation reaction catalyzed by phenolic acid decarboxylase PAD_Cs.

[0037] Figure 4 The β-carboxylation reaction conversion results of phenolic acid decarboxylase PAD_Cs catalyzing different reaction substrates (4-vinylphenol compounds) to synthesize different reaction products under standard β-carboxylation conditions.

[0038] Figure 5This is the HPLC chromatogram of the synthesis of compound 1b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0039] Figure 6 This is the mass spectrum of compound 1b synthesized by phenolic acid decarboxylase PAD_Cs.

[0040] Figure 7 This is the HPLC chromatogram of the synthesis of compound 2b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0041] Figure 8 This is the mass spectrum of compound 2b synthesized by phenolic acid decarboxylase PAD_Cs.

[0042] Fig. 9 This is the HPLC chromatogram of the synthesis of compound 3b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0043] Fig.10 This is the mass spectrum of compound 3b synthesized by phenolic acid decarboxylase PAD_Cs.

[0044] Fig.11 This is the HPLC chromatogram of the synthesis of compound 5b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0045] Fig.12 This is the mass spectrum of compound 5b synthesized by phenolic acid decarboxylase PAD_Cs.

[0046] Fig.13 This is the high performance liquid chromatogram of the synthesis of compound 6b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0047] Fig.14 This is the mass spectrum of compound 6b synthesized by phenolic acid decarboxylase PAD_Cs.

[0048] Fig.15 This is the HPLC chromatogram of the synthesis of compound 7b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0049] Fig.16 This is the mass spectrum of compound 7b synthesized by phenolic acid decarboxylase PAD_Cs.

[0050] Fig.17 This is the HPLC chromatogram of the synthesis of compound 8b catalyzed by phenolic acid decarboxylase PAD_Cs.

[0051] Fig.18 This is the mass spectrum of compound 8b synthesized by phenolic acid decarboxylase PAD_Cs.

[0052] Fig.19 This is the effect of different substrate (1a, i.e., p-hydroxystyrene) concentrations on the yield of p-coumaric acid synthesized by PAD_Cs phenolic acid decarboxylase β-carboxylation. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0054] 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.

[0055] Example 1: Construction of a recombinant strain expressing phenolic acid decarboxylase based on Escherichia coli In order to improve the soluble expression of protein, the Clostridium sp. DSM8431 strain phenolic acid decarboxylase PAD_Cs sequence was codon optimized, and the optimized nucleotide sequence is shown in SEQ ID NO: 2. Ncol I restriction site (5'-TAACTTTAAGAAGGAGATATACCATGGCGATGAAAAACAAAA-3') and XOt The gene of phenolic acid decarboxylase PAD_Cs was amplified by using the reverse primer (5'-TCAGTGGTGGTGGTGGTGGTGCTCGAGTTTAATTTTTTTATA-3') with the restriction endonuclease I. The amplified DNA fragment was combined with the vector pET28a(+) using restriction endonucleases Ncol I and XOt I (purchased from Bao Ri Yi Biotechnology Co., Ltd.) was used for double enzyme digestion. The enzyme digestion reaction was carried out in a 50 μL system and was prepared and reacted according to the instructions of the DNA restriction endonuclease. After the enzyme digestion was completed, the DNA fragments were recovered by gel cutting and connected with the enzyme-cut vector at a material ratio of 3:1. The connection system was prepared according to 10 μL, and T4 ligase was used to catalyze the connection reaction. The connection was carried out at 16°C overnight to obtain the recombinant plasmid pET28a-PAD_Cs. The above synthesis process was completed by Azenta Life Sciences, and the recombinant plasmid pET28a-PAD_Cs was stored in E. coli DH5α.

[0056] According to the instructions of the "yPrp plasmid DNA small-scale extraction kit", the plasmid pET-28a-PAD_Cs was extracted. The recombinant plasmid pET28a-PAD_Cs was transformed into Escherichia coli using the heat shock method. E. coli BL21(DE3) competent cells were plated on LB plates containing 100 μg / mL kanamycin sulfate and cultured at 37 °C for 14-16 h. The sequencing results were verified by sequencing (completed by Anhui General Biotechnology Co., Ltd.) to obtain the recombinant strain. E. coli BL21(DE3)-PAD_Cs.

[0057] Example 2: Expression and purification of phenolic acid decarboxylase PAD_Cs 1. Inducible expression 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 seed solution. The seed solution was inoculated into 50 mL of fresh TB liquid medium at an inoculum volume of 2% v / v and cultured at 37°C and 180 rpm until OD 600 When the pH value was between 0.6 and 1.0, the cells were taken out and cooled in an ice-water bath for 5 min. The inducer IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1 mmol / L. The expression was induced at 16 °C and 150 rpm for 20 h to obtain the fermentation broth.

[0058] 2. Purification of phenolic acid decarboxylase PAD_Cs Take the fermentation liquid of induced expression, centrifuge at 12000 rpm for 20 min, discard the supernatant, then resuspend and wash the bacteria with 20 mM Tris-HCl (pH7.5) buffer, centrifuge at 12000 rpm for 20 min, discard the supernatant, resuspend again with buffer, and ultrasonically disrupt. Centrifuge the ultrasonic disrupted liquid at 12000 rpm for 20 min, and take the supernatant (i.e. crude enzyme solution) for subsequent SDS-PAGE electrophoresis detection.

[0059] The crude enzyme solution was filtered through a 0.22 μm filter membrane. The nickel column was flushed with Buffer A (20 mM Tris-HCl, pH 7.5) at a flow rate of 2 mL / min until equilibrium. The protein sample was injected into the injection loop with a syringe, and the penetration peak protein of the sample was collected. The nickel column was flushed with Buffer A (20 mM Tris-HCl, pH 7.5) again until no protein was eluted. The gradient elution method was used, and the nickel column was flushed with at least 5 volumes of Buffer B (20 mM Tris-HCl, 500 mM imidazole, pH 7.5) for each gradient, and the absorption peak protein of each gradient was collected until no protein was eluted. The protein solution purified by the nickel column was replaced with 20 mM Tris-HCl (pH 7.5) buffer using a desalting column pre-installed by GE to remove the imidazole in the protein solution. The collected protein solutions of different gradients were verified by SDS-PAGE. The results showed that the target protein eluted with 15% Buffer B had a higher purity. Figure 1Lane 4 in the figure shows the pET-28a-PAD_Cs band eluted with 15% Buffer B.

[0060] The SDS-PAGE electrophoresis conditions were as follows: the concentration of the concentrated gel was 4%, and the concentration of the separation gel was 12.5%; the sample and the loading buffer were mixed in a ratio of 3:1, and the sample was reacted in a boiling water bath for 5 min before loading electrophoresis; the initial voltage of the electrophoresis instrument was set to 120 V, and the voltage was increased to 230 V when the sample moved to the separation gel, and the electrophoresis was terminated when the sample moved to the bottom of the electrophoresis tank.

[0061] The results of SDS-PAGE electrophoresis analysis of phenolic acid decarboxylase PAD_Cs are shown in Figure 1 As shown, it can be seen 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 was successfully induced to express.

[0062] Example 3: Optimization of the decarboxylation reaction of phenolic acid decarboxylase PAD_Cs to produce p-hydroxystyrene 1. Optimization of the optimal temperature for the decarboxylation reaction of phenolic acid decarboxylase After the purified phenolic acid decarboxylase PAD_Cs enzyme solution was diluted to 0.1 mg / mL, 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 pH 6.0 citric acid buffer were added to 1 mL of the reaction system. After mixing evenly, the mixture was reacted at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, and 65 ℃ for 5 min, and then 2 mL of acetonitrile was added to terminate the reaction to obtain a mixture. The mixture was centrifuged at 4 ℃ and 12,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC) to determine the enzyme activity at different reaction temperatures. The highest enzyme activity at the above reaction temperature was used as the control, and the relative enzyme activity was calculated in turn, and the curve of enzyme activity changing with temperature was drawn.

[0063] The preparation of the p-coumaric acid solution is as follows: add 8.21 g of p-coumaric acid accurately weighed into a certain amount of pure water, adjust the pH to 7.0 with NaOH solution, stir with a magnetic stirrer and monitor the pH of the solution with a pH meter, and after the p-coumaric acid powder is completely dissolved, adjust the volume to 1 L, at which time the final concentration of the p-coumaric acid solution is 50 mM.

[0064] 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.

[0065] The results are as follows Figure 2 As shown, the optimum 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.

[0066] From snake moss Conocephalum japonicum Phenolic acid decarboxylase CjPAD from Lactobacillus Lactobacillus brevis RM84 phenolic acid decarboxylase L. brevis PAD, from Bacillus amyloliquefaciens Bacillus amyloliquefaciens The phenolic acid decarboxylase BaPAD-Q-426 of Q-426 was used as a control. The results showed that the optimum temperature of phenolic acid decarboxylase CjPAD was 25°C, the optimum temperature of phenolic acid decarboxylase L. brevis PAD was 30°C, and the optimum temperature of phenolic acid decarboxylase BaPAD-Q-426 was 35°C. Further literature review revealed that the optimum temperature of phenolic acid decarboxylase is generally 20~40°C, which is much lower than the optimum temperature of phenolic acid decarboxylase PAD_Cs. A higher optimum temperature means that phenolic acid decarboxylase PAD_Cs has better catalytic performance at higher temperatures.

[0067] 2. Determination of the optimal pH for the decarboxylation reaction of phenolic acid decarboxylase After the purified phenolic acid decarboxylase PAD_Cs enzyme solution was diluted to 0.1 mg / mL, 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 pH 3.0-9.0 buffer (50 mM pH 3.0-5.5 citric acid-sodium citrate buffer, 50 mM pH 5.5-8.0 PB buffer, 50 mM pH 8.0-9.0 Tris-HCl buffer) were added to 1 mL of the reaction system, mixed evenly, reacted at 50 °C for 5 min, and then 2 mL of acetonitrile was added to terminate the reaction to obtain a mixture. The mixture was centrifuged at 4 °C and 12,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC) to determine the enzyme activity under different pH citric acid buffers. The highest enzyme activity at different pH values ​​was used as a control, and the relative enzyme activity was calculated in turn, and a curve of enzyme activity changing with the pH value of the citric acid buffer was drawn. The definition of enzyme activity is the same as above.

[0068] The results are as follows Figure 3 As shown, the optimum pH of phenolic acid decarboxylase PAD_Cs is 5.0. The enzyme activity decreases with the increase or decrease of pH, and almost loses its activity when pH ≤ 4 and ≥ 9.

[0069] Example 4: Determination and optimization of the carboxylation reaction of phenolic acid decarboxylase PAD_Cs 1. Preparation of Lyophilized Whole Cells of Recombinant Strain The fermentation liquid obtained by inducing the expression of the recombinant strain was centrifuged at 4 ℃ and 12000 rpm for 10 min; the centrifugal supernatant was discarded, the bacterial cells were collected, and an appropriate amount of pure water was added to resuspend the precipitated bacterial cells, and the bacterial cells were collected by centrifugation again, and repeated 2~3 times; the precipitated bacterial cells were placed in a -80 ℃ ultra-low temperature refrigerator for pre-freezing for 12 h. The pre-frozen precipitated bacterial cells were quickly placed in a freeze dryer for freeze-drying for 12~16 h to obtain the recombinant strain freeze-dried bacterial powder, that is, the recombinant strain freeze-dried whole cells.

[0070] 2. β-Carboxylation reaction of different substrates In order to investigate whether phenolic acid decarboxylase PAD_Cs has the ability to catalyze carboxylation reaction under standard β-carboxylation reaction conditions, different 4-vinylphenol compounds (1a~8a) substituted with groups with different electronic characteristics and steric hindrance were used as substrates to determine the conversion rate of carboxylation reaction. The reaction product was compared with the standard in HPLC and combined with mass spectrometry to determine whether the reaction product was generated. The reaction conversion rate was calculated by drawing the standard curve of the reaction product.

[0071] The specific determination process is as follows: In a 1 mL reaction system, 20 mg / mL of lyophilized whole cells of the recombinant strain were resuspended in 100 mM pH 5.5 phosphate buffer, shaken and hydrated at 600 rpm and 30°C for 30 min, and the substrate (1a~8a) was added directly or added from a substrate solution dissolved in acetonitrile to make the final concentration of the substrate in the reaction system 10 mM, and 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 a reaction solution. The above reaction system was carried out in a sealed glass bottle with a lid and CO2 (the bottle mouth was wrapped with a sealing film to prevent CO2 from escaping). Empty host cells without overexpressed phenolic acid decarboxylase were used as negative controls.

[0072] The reaction solution was centrifuged at 13000 rpm for 15 min, the supernatant was taken, and each 100 μL reaction solution was diluted with 1 mL of water / acetonitrile mixture (v / v 1:1), and 3% v / v trifluoroacetic acid (based on water / acetonitrile mixture, i.e., 30 μL) was added. After incubation at room temperature for 5 min, the reaction was centrifuged at 13000 rpm for 15 min, and the β-carboxylation reaction conversion was analyzed by high performance liquid chromatography. The reaction product was identified by comparison with the standard and by mass spectrometry.

[0073] The high performance liquid chromatography detection method is as follows: All liquid chromatography measurements were performed 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.

[0074] (1) Compounds 1a, 1b, 2a, 2b, 4a, 4b, 8a, and 8b were detected spectrophotometrically at 280 and 320 nm using 0.1% H2O / TFA (0.1%) and 0.1% MeCN / TFA (40:60, v / v) as the mobile phase, respectively.

[0075] (2) Compounds 3a, 3b, 7a, and 7b were detected spectrophotometrically at 280 and 320 nm using 0.1% H2O / TFA (0.1%) and 0.1% (20:80, v / v) as the mobile phase, respectively.

[0076] (3) Compounds 5a, 5b, 6a, and 6b were detected spectrophotometrically at 270 and 320 nm using 0.1% H2O / TFA (0.1%) and 0.1% (20:80, v / v) as the mobile phase, respectively.

[0077] In the HPLC detection of β-carboxylation reaction of different substrates, the concentration of the reaction product was calculated based on the detection data at 320 nm ultraviolet wavelength. Figure 4 The β-carboxylation reaction conversion rate results 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, and 17 are HPLC chromatograms of the synthesis of compounds 1b~3b, 5b~8b catalyzed by phenolic acid decarboxylase PAD_Cs; Figure 6 , 8, 10, 12, 14, 16, and 18 are the mass spectra of compounds 1b~3b, 5b~8b catalyzed by phenolic acid decarboxylase PAD_Cs. As shown in the figure, 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 and alkoxy groups and halogens. In the carboxylation reaction, the conversion rate of phenolic acid decarboxylase PAD_Cs for the reaction substrate 2a (2-methoxy-4-vinylphenol) is the highest, which is 26%, followed by the conversion rate of the reaction substrate 1a (4-vinylphenol, i.e., p-hydroxypropylvinyl) with a conversion rate of 20%; but the reaction for the reaction substrate 4a (2-methyl-4-vinylphenol) is not obvious, with a conversion rate of <1%.

[0078] Lactobacillus plantarum Lactobacillus plantarum PAD Lp and Lactococcus lactis Lactoccocus lactis Phenolic acid decarboxylase PAD LI from Bacillus amyloliquefaciens Bacillus amyloliquefaciens The phenolic acid decarboxylase PAD_Ba of the present invention was used as a control to investigate the conversion rates of different substrate spectra. The results showed that the conversion rate of phenolic acid decarboxylase PAD Lp in synthesizing p-coumaric acid was only 2%; the conversion rate of phenolic acid decarboxylase PAD LI in synthesizing p-coumaric acid was only 3%, and the conversion rate of ferulic acid was only 2%; the conversion rate of phenolic acid decarboxylase PAD_Ba in synthesizing p-coumaric acid was 18%, indicating that the yield of phenolic acid decarboxylase PAD_Cs for biologically active phenolic acid compounds such as p-coumaric acid and ferulic acid is at the forefront among similar enzymes. In addition, in the synthesis of p-coumaric acid, the above-mentioned phenolic acid decarboxylases all require acetonitrile with a volume fraction of more than 20% in the reaction system. When the phenolic acid decarboxylase PAD Cs of the present invention synthesizes p-coumaric acid, the specific acetonitrile volume fraction required is ≤12% v / v, that is, only a lower volume fraction of acetonitrile is required to obtain a 20% p-coumaric acid yield. In summary, phenolic acid decarboxylase PAD_Cs is a good biocatalytic carboxylation tool with great potential for CO2 fixation.

[0079] 3. Effect of different substrate concentrations on the β-carboxylation reaction of phenolic acid decarboxylase PAD_Cs on coumaric acid production In a 1 mL reaction system, 20 mg / mL of the recombinant strain freeze-dried whole cells were resuspended in 100 mM pH 5.5 phosphate buffer, shaken and hydrated at 600 rpm and 30°C for 30 min, and compound 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 the cosolvents 0-20% v / v acetonitrile and 3 MKHCO3 were added, and the reaction was carried out at 30°C and 600 rpm for 24 h to obtain a reaction solution. The above reaction system was carried out in a sealed glass bottle with a lid and CO2 (the bottle mouth was wrapped with a sealing film to prevent CO2 from escaping). The above process was carried out according to the carboxylation reaction determination process, the yield of the carboxylation product p-coumaric acid was determined, and a curve of the yield changing with the substrate concentration was drawn.

[0080] The results are as follows Fig.19 It can be seen that at a p-hydroxystyrene concentration of 80 mM, the maximum yield of p-coumaric acid can reach 9.7 mM.

[0081] 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 approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant strain expressing phenolic acid decarboxylase, characterized in that: By heterologous expression of the gene encoding phenolic acid decarboxylase PAD_Cs , obtaining the recombinant strain; Wherein, the coding gene of the phenolic acid decarboxylase PAD_Cs , whose nucleotide sequence is shown in SEQ ID NO:

2.

2. The method for constructing the recombinant strain according to claim 1, characterized in that: The gene encoding phenolic acid decarboxylase PAD_Cs The recombinant plasmid is transformed into a host cell to construct the recombinant strain.

3. Use of the recombinant strain according to claim 1 in catalytic synthesis of 4-vinylphenol.

4. The use according to claim 3, characterized in that: The catalytic synthesis uses 3-(4-hydroxyphenyl) acrylic acid as a substrate and utilizes a recombinant strain induced expression and purified enzyme solution to catalyze the decarboxylation reaction of 3-(4-hydroxyphenyl) acrylic acid to synthesize 4-vinylphenol.

5. The use according to claim 4, characterized in that: The reaction system of the catalytic synthesis is: 10-50 mM 3-(4-hydroxyphenyl)acrylic acid, 0.1-1 mg / mL recombinant strain induced expression, purified enzyme solution, 50 mM pH 3.0-9.0 buffer; the conditions of the catalytic synthesis are: reaction at 30-70°C for 5-15 min.

6. Use of the recombinant strain according to claim 1 in catalytic synthesis of 3-(4-hydroxyphenyl) acrylic acid compounds.

7. The use according to claim 6, characterized in that: The catalytic synthesis uses 4-vinylphenol compounds as substrates and bicarbonate as a carboxyl source, and utilizes freeze-dried whole cells of the recombinant strain to catalyze the carboxylation reaction of the 4-vinylphenol compounds to synthesize 3-(4-hydroxyphenyl) acrylic acid compounds.

8. The use according to claim 7, characterized in that: The 4-vinylphenol compound has the structural formula: ; The 3-(4-hydroxyphenyl) acrylic acid compound has the structural formula: ; Among them, the R 1 Any one selected from hydrogen, methyl, methoxy, ethoxy, chlorine, and bromine, wherein R 2 Any one selected from hydrogen, methyl and methoxy.

9. The use according to claim 7, characterized in that: The catalytic synthesis 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; the conditions of the catalytic synthesis are: 30-40°C, 200-1000 rpm, and reaction time for 12-24 h.

10. The use according to claim 7 or 9, characterized in that: The freeze-dried whole cells of the recombinant strain are obtained by centrifuging the fermentation broth obtained by inducing expression of the recombinant strain, collecting the bacterial cells, washing them with pure water 2 to 3 times, pre-freezing the bacterial cells at -80°C for 12 hours, and then rapidly freeze-drying them for 12 to 16 hours.

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