Phenolic acid decarboxylase mutant and application thereof

Abstract

The invention relates to the field of gene engineering, in particular to a phenolic acid decarboxylase mutant and application thereof. According to the invention, tryptophan at the 30th site of phenolic acid decarboxylase PADCs from Clostridium sp.DSM 8431 is mutated into leucine, and tyrosine at the 136th site of the phenolic acid decarboxylase PADCs is mutated into tryptophan, so that the phenolic acid decarboxylase mutant is obtained. The phenolic acid decarboxylase mutant can catalyze decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; and hydrocarbonate can also be used as a carboxyl source to catalyze carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid. The phenolic acid decarboxylase PADCs has the enzyme activity characteristics of excellent temperature stability and acetonitrile stability, still retains more than 80% of decarboxylase activity after being incubated for 2 hours at 55 DEG C, still retains about 36% of decarboxylase activity after being incubated for 2 hours in acetonitrile with the volume fraction of 50%, can remarkably improve the yield of p-coumaric acid, can reach 13.05 mM when the substrate concentration is 80 mM, and is improved by 74% compared with original phenolic acid decarboxylase PADCs; the method has a wide industrial application prospect and CO2 fixation potential.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a phenolic acid decarboxylase mutant and application thereof. Background Art

[0002] p-Coumaric acid ( p p-Coumaric acid (CA) is a plant-derived secondary metabolite belonging to the class of phenolic compounds. It is widely distributed in the plant kingdom, mainly found in fruits, vegetables, and cereals. p-Coumaric acid and its derivatives have various biological activities such as antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, and anti-melanogenesis. Due to its potent free radical scavenging activity, it can alleviate the adverse effects of various diseases, including arthritis, neurological disorders, and cardiovascular diseases.

[0003] In the prior art, phenolic acid decarboxylase has been widely studied for the bioproduction of vinylphenol. In order to further expand the prospects of phenolic acid decarboxylase in industrial production, it is extremely important to improve its stability. For example, by using protein NC-terminal engineering, the N-terminal of the amino acid sequence of phenolic acid decarboxylase from Bacillus amyloliquefaciens ZJH-01 was extended, which improved the enzyme's alkali resistance and heat resistance; by combining ancestral sequence reconstruction and solvent engineering, the enzyme's thermal stability and the solubility of the substrate p-coumaric acid in the reaction system were improved. Existing protein modification research on phenolic acid decarboxylase is mainly aimed at the decarboxylation activity of the enzyme, but it involves the carboxylation activity of phenolic acid decarboxylase and the fixation of CO. 2 Therefore, further research on phenolic acid decarboxylase with carboxylation activity is still of great value. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a phenolic acid decarboxylase mutant in view of the deficiencies in the prior art.

[0005] The technical problem that the present invention needs to solve is to provide the application of the above-mentioned phenolic acid decarboxylase mutant.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A phenolic acid decarboxylase mutant is obtained by mutating the 30th tryptophan to leucine (W30L) and the 136th tyrosine to tryptophan (Y136W) of the phenolic acid decarboxylase PAD_Cs.

[0007] The amino acid sequence of the phenolic acid decarboxylase PAD_Cs is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence after codon optimization is shown in SEQ ID NO: 2.

[0008] Wherein, the phenolic acid decarboxylase PAD_Cs is derived from Clostridium Clostridiumsp. DSM 8431.

[0009] The phenolic acid decarboxylase mutant is obtained by first finding two potential amino acid active sites W30 and Y136 based on computer-aided design, then performing saturation mutation on these two sites, preferably obtaining two phenolic acid decarboxylase single-point mutants W30L and Y136W, and then performing combined mutation on W30L and Y136W.

[0010] Specifically, the amino acid sequence of the phenolic acid decarboxylase mutant is shown in SEQ ID NO:7.

[0011] A DNA molecule encoding the phenolic acid decarboxylase mutant is also within the scope of protection of the present invention.

[0012] Specifically, the coding gene of the DNA molecule is shown in SEQ ID NO:8.

[0013] A recombinant expression vector containing the coding gene of the DNA molecule is also within the scope of protection of the present invention.

[0014] A recombinant strain is also within the scope of protection of the present invention.

[0015] Specifically, the recombinant strain is obtained by introducing the coding gene of the DNA molecule into a host cell, or by introducing the coding gene of the DNA molecule into a host cell via the recombinant expression vector.

[0016] Wherein, the host cell is Escherichia coli.

[0017] In some embodiments of the present invention, the host cell is E. coli BL21(DE3).

[0018] The application of the phenolic acid decarboxylase mutant in the catalytic preparation of p-hydroxystyrene is also within the scope of protection of the present invention.

[0019] The catalysis uses p-coumaric acid as a substrate and utilizes a phenolic acid decarboxylase mutant to catalyze a decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene.

[0020] Specifically, the catalysis reaction system is: 10-50 mM p-coumaric acid, 0.1-1 mg / mL phenolic acid decarboxylase mutant, 50 mM pH 3.0-5.5 citric acid-sodium citrate buffer.

[0021] Specifically, the catalysis conditions are as follows: reaction at 30-80° C. for 5-15 min.

[0022] In some embodiments of the present invention, the phenolic acid decarboxylase mutant has excellent enzyme activity characteristics of temperature stability and acetonitrile stability, retaining more than 80% of the decarboxylase activity after incubation at 55°C for 2 h, and retaining about 36% of the decarboxylation activity after incubation in 50% volume fraction acetonitrile for 2 h.

[0023] The application of the phenolic acid decarboxylase mutant in the catalytic preparation of p-coumaric acid is also within the scope of protection of the present invention.

[0024] The catalysis uses p-hydroxystyrene as a substrate and bicarbonate as a carboxyl source, and utilizes a phenolic acid decarboxylase mutant to catalyze a carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid.

[0025] Specifically, the catalysis, the catalytic reaction in CO 2 The catalytic reaction system was: 20-40 mg / mL freeze-dried whole cells of phenolic acid decarboxylase mutant, 100 mM phosphate buffer at pH 5.5-7.0, 10-100 mM p-hydroxystyrene, 0-20% v / v acetonitrile, 1-3 M KHCO 3 .

[0026] Specifically, the catalysis conditions are: 30-40° C., 200-1000 rpm, and reaction time of 12-24 h.

[0027] In some embodiments of the present invention, the phenolic acid decarboxylase mutant can significantly increase the yield of p-coumaric acid. When the substrate concentration is 80 mM, the yield can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs.

[0028] Beneficial effects: The present invention is carried out by Clostridium The phenolic acid decarboxylase PAD_Cs from sp. DSM 8431 was obtained by mutating the tryptophan at position 30 to leucine (W30L) and the tyrosine at position 136 to tryptophan (Y136W). The mutant phenolic acid decarboxylase can catalyze the decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; it can also catalyze the carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid using bicarbonate as a carboxyl source. It has excellent enzyme activity characteristics of temperature stability and acetonitrile stability. It still retains more than 80% of the decarboxylase activity after incubation at 55℃ for 2 h, and still retains about 36% of the decarboxylation activity after incubation in 50% volume fraction acetonitrile for 2 h. It can also significantly increase the yield of p-coumaric acid. When the substrate concentration is 80 mM, the yield can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs. It has broad industrial application prospects and carbon dioxide fixation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 The SDS-PAGE diagram of phenolic acid decarboxylase PAD_Cs and its single point and combined mutants. Among them, M: Marker; 1: phenolic acid decarboxylase PAD_Cs; 2: phenolic acid decarboxylase single point mutant W30L; 3: phenolic acid decarboxylase single point mutant Y136W; 4: phenolic acid decarboxylase combined mutant W30L / Y136W.

[0031] Figure 2 Thermal stability analysis of phenolic acid decarboxylase PAD_Cs and its mutant W30L / Y136W.

[0032] Figure 3 Analysis of the acetonitrile stability of phenolic acid decarboxylase PAD_Cs and its mutant W30L / Y136W.

[0033] Figure 4 The effect of different substrate (p-hydroxystyrene) concentrations on the production of p-coumaric acid by the phenolic acid decarboxylase combined mutant W30L / Y136W. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] In the following examples, the high performance liquid chromatography (HPLC) detection method is as follows: All liquid chromatography measurements are performed using an Agilent HPLC (Model-1200, phase) equipped with C18 (Agilent 5 HC-C18, 250×4.6 mm, 5 μm). The column temperature is 24°C, the flow rate is 1 mL / min, the program runs for 10 min, and the mobile phase and UV detection conditions are: 0.1% H 2 O / TFA and 0.1% MeCN / TFA (40∶60, v / v) were used as mobile phases, and spectrophotometric detection was performed at 280 nm and 320 nm. The concentration of the reaction product was calculated based on the detection data of the ultraviolet wavelength of 280 nm for the decarboxylation reaction, and the concentration of the reaction product was calculated based on the detection data of the ultraviolet wavelength of 320 nm for the β-carboxylation reaction.

[0037] Example 1: Determination of mutation site of phenolic acid decarboxylase mutant Download the clostridium from UniProt database Clostridium sp. DSM 8431 strain, and docked the substrate p-hydroxystyrene to the active pocket. The amino acids within 4 Å of the active site were selected, and combined with the multiple sequence alignment of the reported phenolic acid decarboxylases with carboxylation activity, it was found that the W30 site was quite different, while the rest of the amino acids were highly conserved. In addition, based on the sequence conservation, it was found that at the Y136 site, the phenolic acid decarboxylase PAD_Cs was significantly different from the other compared phenolic acid decarboxylases.

[0038] Example 2: Construction, expression and screening of single-point mutants of phenolic acid decarboxylase 1. Construction of plasmid pET-28a-PAD_Cs The codon-optimized sequence of the target phenolic acid decarboxylase (PAD_Cs) gene (WP_090014602.1) (shown in SEQ ID NO: 2) was submitted to Azenta Life Sciences, and the plasmid pET-28a(+) was synthesized using pET-28a-PAD_Cs and stored in E. coli DH5α.

[0039] 2. Construction and expression of single-point mutants of phenolic acid decarboxylase (1) Construction of single-point mutants of phenolic acid decarboxylase Saturation mutations were performed on the two sites, W30 and Y136, respectively. The amino acid W at the 30 site was mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or Y, and the amino acid Y at the 136 site was mutated to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W, respectively. A total of 38 single-point mutants of phenolic acid decarboxylase (W30A, W30C, W30D, W30E, W30F, W30G , W30H, W30I, W30K, W30L, W30M, W30N, W30P, W30Q, W30R, W30S, W30T, W30V, W30Y, Y136A, Y136C, Y136D, Y136E, Y136F, Y136G, Y136H, Y136I, Y136K, Y136L, Y136M, Y136N, Y136P, Y136Q, Y136R, Y136S, Y136T, Y136V, Y136W).

[0040] The specific process is as follows: According to the relevant instructions of the "yPrp Plasmid DNA Mini-Extraction Kit", extract the plasmid pET-28a-PAD_Cs. Using the plasmid pET-28a-PAD_Cs as a template, refer to the Vazyme biological product and operation manual, use the corresponding mutation primer pair, and amplify the site-directed mutation sequence by whole plasmid PCR. Among them, the whole plasmid PCR, its reaction system includes: 12.5μL Phanta high-fidelity enzyme, 1 μL template (plasmid pET-28a-PAD_Cs), 1 μL primer F, 1 μL primer R, ddH 2 O 9.5μL; the reaction parameters are: 95℃ 2min, 95℃ 15 s, 60℃ 15 s, 72℃ extension 3 min, 30 cycles, 72℃ 10 min. The above-mentioned phenolic acid decarboxylase single point mutants W30L and Y136W, when using the corresponding mutant primer pairs to amplify the site-directed mutant sequence by whole plasmid PCR, the sequences of the mutant primer pairs used are shown in Table 1.

[0041] Table 1 Primer pairs for the mutation of phenolic acid decarboxylase single point mutants W30L and Y136W

[0042] The PCR products were used Dpn After the template digestion is completed, the heat shock method is used to transform into Escherichia coli competent cells. E. coli BL21(DE3), and spread on LB agar plates containing 100 μg / mL kanamycin sulfate, and cultured inverted overnight at 37°C. The mutation results of overnight culture were sequenced and verified by Anhui General Biological Company.

[0043] (2) Expression of single-point mutants of phenolic acid decarboxylase The recombinant E. coli with the correctly sequenced single-point mutant of phenolic acid decarboxylase 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 prepare seed solution. The seed solution was then inoculated into fresh 50 mL of LB liquid medium at a 2% v / v inoculation rate 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.

[0044] 3. Screening of single-point mutants of phenolic acid decarboxylase Due to the long carboxylation reaction time and the presence of acetonitrile, an organic co-solvent, in the reaction system, the stability of the enzyme is the key to its continued catalytic effect. Therefore, the conversion rate of the single-point mutant in the presence of 12% and 20% acetonitrile volume fractions in the reaction system was selected as the screening criterion for screening acetonitrile-stable phenolic acid decarboxylase single-point mutants. The specific process is as follows.

[0045] (1) Preparation of freeze-dried whole cells of single-point mutants of phenolic acid decarboxylase The fermentation broth obtained by inducing the expression of the recombinant Escherichia coli of the single-point mutant of phenolic acid decarboxylase 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 to 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 to 16 h, and freeze-dried bacterial powders of different single-point mutants of phenolic acid decarboxylase, namely freeze-dried whole cells of single-point mutants of phenolic acid decarboxylase, were obtained respectively.

[0046] (2) Screening of single-point mutants of phenolic acid decarboxylase The 1 mL reaction system for β-carboxylation reaction was as follows: 20 mg / mL of freeze-dried whole cells of the phenolic acid decarboxylase combination mutants were resuspended in 100 mM phosphate buffer, pH 5.5, and hydrated at 600 rpm, 30°C for 30 min. Then, 10 mM p-hydroxystyrene (with propylene glycol as solvent), 12% v / v or 20% v / v acetonitrile, and 3 M KHCO were added. 3 The reaction was carried out at 30 °C and 600 rpm for 24 h to obtain a reaction solution. 2 Glass bottle (the bottle mouth is wrapped with sealing film to prevent CO 2 escape).

[0047] The reaction solution was centrifuged at 13000 rpm for 15 min, the supernatant was taken, and each 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, i.e., 30 μL) was added. After incubation at room temperature for 5 min, the mixture was centrifuged at 13000 rpm for 15 min, and the β-carboxylation reaction conversion rate was analyzed by reversed-phase high performance liquid chromatography.

[0048] The results are shown in Table 2. It can be seen from the table that at a volume fraction of 12% acetonitrile, most of the phenolic acid decarboxylase single point mutants have good conversion rates. When the volume fraction of acetonitrile is increased to 20%, the phenolic acid decarboxylase single point mutants W30L and Y136W show the best conversion rates, with W30L being 3.71% and Y136W being 2.65%, indicating that these two single point mutants retain significant carboxylation activity and have high acetonitrile stability.

[0049] Table 2 Conversion rate of β-carboxylation reaction using different single-point mutants of phenolic acid decarboxylase

[0050] Example 3: Construction, expression and purification of the phenolic acid decarboxylase combined mutant W30L / Y136W The plasmid of the single point mutant W30L of phenolic acid decarboxylase was used as a template, and the Y136W-F and Y136W-R mutant primer pairs were used for PCR amplification. The PCR amplification process, PCR product digestion and transformation, and induced expression of the combined mutant W30L / Y136W bacteria were the same as in Example 2.

[0051] 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, which is the crude enzyme solution.

[0052] 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 each gradient was washed with at least 5 volumes of 15% Buffer B (20 mM Tris-HCl, 500 mM imidazole, pH 7.5) to flush the nickel column, and the absorption peak protein during elution was collected until no protein was eluted. The protein solution purified by the nickel column was replaced with the 20 mM Tris-HCl (pH 7.5) buffer using a desalting column pre-installed by GE to remove the imidazole in the protein solution, and finally the purified enzyme solution was obtained.

[0053] The purified enzyme solutions of phenolic acid decarboxylase PAD_Cs, phenolic acid decarboxylase single point mutants W30L, Y136W and phenolic acid decarboxylase combined mutants W30L / Y136W were subjected to SDS-PAGE electrophoresis. Figure 1 As shown, the molecular weight of the target protein is 21 kDa, and each lane in the electrophoresis diagram has an obvious band at 21 kDa, indicating that the target protein is successfully expressed in each mutant.

[0054] Example 4: Decarboxylase activity assay of the phenolic acid decarboxylase combined mutant W30L / Y136W Using p-coumaric acid solution as substrate, p-hydroxystyrene was catalyzed to form the decarboxylase activity of the phenolic acid decarboxylase combination mutant W30L / Y136W after incubation at different temperatures and incubation with different volume fractions of acetonitrile, in order to consider the enzymatic properties of the phenolic acid decarboxylase combination mutant.

[0055] The definition of enzyme activity unit is: 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.

[0056] Among them, the preparation method of p-coumaric acid solution is as follows: accurately weigh 8.21 g of p-coumaric acid and place it in 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, and after the p-coumaric acid powder is completely dissolved, adjust the volume to 1 L, and the final concentration of the p-coumaric acid solution is 50 mM.

[0057] 1. Temperature stability The purified enzyme solution of the phenolic acid decarboxylase combined mutant W30L / Y136W was diluted to 0.1 mg / mL and incubated in a water bath at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, and 70 ℃ for 2 h. In a 1 mL reaction system, a final concentration of 50 mM p-coumaric acid solution, W30L / Y136W enzyme solution incubated at different temperatures, and 50 mM citric acid-sodium citrate buffer (pH 5.0) were added and reacted at 60 ℃ for 5 min. Then 2 mL of acetonitrile was added to terminate the reaction. The reaction mixture was centrifuged at 4 ℃ and 12,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC). Phenolic acid decarboxylase PAD_Cs (represented by WT in the figure) was used as a control.

[0058] The initial enzyme activity without temperature incubation was recorded as 100%, and the relative enzyme activities of the original phenolic acid decarboxylase PAD_Cs and the combined mutants after incubation at each temperature were calculated, and the change curves of the residual enzyme activity under different incubation conditions were plotted. Figure 2As shown in the results, the temperature stability of the combined mutant W30L / Y136W was significantly improved compared with the original phenolic acid decarboxylase PAD_Cs. The phenolic acid decarboxylase PAD_Cs could only retain 80% of its activity when the incubation temperature was less than 35 ℃. After incubation at ≥50 ℃ for 2 h, the phenolic acid decarboxylase PAD_Cs almost lost its activity. However, when the combined mutant W30L / Y136W had an insignificant decrease in enzyme activity during the incubation temperature of 30-55 ℃, it still retained more than 80% of its activity after incubation at 55 ℃ for 2 h. In addition, the decarboxylation specific enzyme activity of the combined mutant increased from 177 IU / mg of the original phenolic acid decarboxylase PAD_Cs to 335 IU / mg, which was about 1.9 times that of the original phenolic acid decarboxylase PAD_Cs.

[0059] 2. Acetonitrile stability The purified enzyme solution of the phenolic acid decarboxylase combined mutant W30L / Y136W was diluted to 0.1 mg / mL and incubated at 4 ℃ on ice for 2 h in different volume fractions of acetonitrile (10% v / v, 20% v / v, 30% v / v, 40% v / v, 50% v / v). In a 1 mL reaction system, a final concentration of 50 mM p-coumaric acid solution, W30L / Y136W enzyme solution incubated with different volume fractions of acetonitrile, and 50 mM citric acid-sodium citrate buffer (pH 5.0) were added and reacted at 60 ℃ for 5 min. Then 2 mL of acetonitrile was added to terminate the reaction. The reaction mixture was centrifuged at 4 ℃ and 12,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC). Phenolic acid decarboxylase PAD_Cs (represented by WT in the figure) was used as a control.

[0060] The initial enzyme activity without incubation in acetonitrile was recorded as 100%, and the residual enzyme activity curve under incubation conditions with different volume fractions of acetonitrile was calculated. Figure 3 As shown in the results, the stability of mutant W30L / Y136W in acetonitrile was significantly improved compared with the original phenolic acid decarboxylase PAD_Cs. After incubation for 2 h at 10-50% acetonitrile volume fraction, the residual enzyme activity of W30L / Y136W was higher than that of phenolic acid decarboxylase PAD_Cs. When incubated in 50% volume fraction acetonitrile, W30L / Y136W still retained about 36% of its activity, while the original enzyme PAD_Cs only had about 16% of its activity. In addition, the specific enzyme activity of the combined mutant in 50% volume fraction acetonitrile was still 131 IU / mg, while the original phenolic acid decarboxylase PAD_Cs was only 30 IU / mg. The specific enzyme activity of the combined mutant was 4.36 times that of the original phenolic acid decarboxylase PAD_Cs.

[0061] Example 5: β-Carboxylation Reaction of Phenolic Acid Decarboxylase Combined Mutant W30L / Y136W (Catalytic Preparation of p-Coumaric Acid) According to the preparation method of freeze-dried whole cells of the phenolic acid decarboxylase single point mutant in Example 1, freeze-dried whole cells of the phenolic acid decarboxylase combination mutant W30L / Y136W were prepared.

[0062] The 1 mL reaction system for the β-carboxylation reaction was as follows: 20 mg / mL of freeze-dried whole cells of the phenolic acid decarboxylase combination mutant were resuspended in 100 mM pH 5.5 phosphate buffer, shaken at 600 rpm and 30°C for 30 min, and then different concentrations of p-hydroxystyrene (20 mM, 40 mM, 60 mM, 80 mM, 100 mM, with propylene glycol as solvent), 0-20% v / v acetonitrile, and 3 M KHCO were added. 3 The reaction was carried out at 30 °C and 600 rpm for 24 h to obtain a reaction solution. 2 Glass bottle (the bottle mouth is wrapped with sealing film to prevent CO 2 Phenolic acid decarboxylase PAD_Cs (indicated by WT in the figure) was used as a control.

[0063] The reaction solution was centrifuged at 13000 rpm for 15 min, the supernatant was taken, and 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, i.e., 30 μL) was added. After incubation at room temperature for 5 minutes, the reaction solution was centrifuged at 13000 rpm for 15 min, and the ultraviolet wavelength data at 320 nm was detected by reversed-phase high performance liquid chromatography to calculate the concentration of the reaction product (i.e., p-coumaric acid) at different substrate concentrations.

[0064] The results are as follows Figure 4 As shown in the results, the combined mutant W30L / Y136W can significantly increase the production of p-coumaric acid. When the substrate concentration is 80 mM, the production can reach 13.05 mM, which is 74% higher than that of the original phenolic acid decarboxylase PAD_Cs. Even at a substrate concentration of 100 mM, which inhibits the β-carboxylation reaction, the p-coumaric acid production of the combined mutant W30L / Y136W is still significantly higher than that of the original phenolic acid decarboxylase PAD_Cs, indicating that the combined mutant has broad industrial application prospects and carbon dioxide fixation potential.

[0065] The present invention provides a phenolic acid decarboxylase mutant and its application ideas and methods. There are many methods and ways 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 regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A phenolic acid decarboxylase mutant, characterized in that: The phenolic acid decarboxylase mutant is obtained by mutating the tryptophan at position 30 of the phenolic acid decarboxylase PAD_Cs to leucine and mutating the tyrosine at position 136 to tryptophan; The amino acid sequence of the phenolic acid decarboxylase PAD_Cs is shown in SEQ ID NO:

1.

2. The phenolic acid decarboxylase mutant according to claim 1, characterized in that The amino acid sequence of the phenolic acid decarboxylase mutant is shown in SEQ ID NO:

7.

3. A DNA molecule, characterized in that The DNA molecule encodes the phenolic acid decarboxylase mutant according to claim 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene of the DNA molecule according to claim 3.

5. A recombinant strain, characterized in that: The recombinant strain is obtained by introducing the coding gene of the DNA molecule of claim 3 into a host cell, or by introducing the coding gene of the DNA molecule of claim 3 into a host cell via the recombinant expression vector of claim 4.

6. Use of the phenolic acid decarboxylase mutant according to claim 1 or 2 in catalyzing the preparation of p-hydroxystyrene.

7. The use according to claim 6, characterized in that: The catalysis uses p-coumaric acid as a substrate and utilizes a phenolic acid decarboxylase mutant to catalyze a decarboxylation reaction of p-coumaric acid to synthesize p-hydroxystyrene; Wherein, the catalysis, the catalytic reaction system is: 10~50 mM p-coumaric acid, 0.1~1 mg / mL phenolic acid decarboxylase mutant, 50 mM pH 3.0~5.5 citric acid-sodium citrate buffer; the catalysis, the catalytic conditions are: reaction at 30~80°C for 5~15 min.

8. Use of the phenolic acid decarboxylase mutant according to claim 1 or 2 in catalyzing the preparation of p-coumaric acid.

9. The use according to claim 6, characterized in that: The catalysis uses p-hydroxystyrene as a substrate and bicarbonate as a carboxyl source, and utilizes a phenolic acid decarboxylase mutant to catalyze a carboxylation reaction of p-hydroxystyrene to synthesize p-coumaric acid.

10. The use according to claim 9, characterized in that: The catalysis is carried out in a CO2 environment, and the catalytic reaction system is: 20-40 mg / mL freeze-dried whole cells of phenolic acid decarboxylase mutants, 100 mM phosphate buffer with a pH of 5.5-7.0, 10-100 mM p-hydroxystyrene, 0-20% v / v acetonitrile, and 1-3 M KHCO3; the catalysis conditions are: 30-40°C, 200-1000 rpm, and reaction time for 12-24 h.

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