An engineered bacterium that efficiently supplies the cofactor prFMN of ferulic acid decarboxylase and its construction method and application

Abstract

The invention discloses an engineering bacterium and a construction method and application of a cofactor prFMN for efficiently supplying ferulic acid decarboxylase, and belongs to the fields of genetic engineering technology and fermentation engineering technology. Based on an Escherichia coli engineering strain (SC-2) overexpressing isopropyl transferase UbiX and ferulic acid decarboxylation Fdc, flavin reductase Fre, formate dehydrogenase FDH, dimethylaminopyridine activating enzyme ThiM and riboflavin kinase RFK are further overexpressed to form an engineering strain SC-6, thereby providing sufficient Fdc cofactor prFMN, and using the whole cell of the recombinant engineering bacteria as a catalyst. Compared with SC-2, with cinnamic acid as a substrate (styrene as a decarboxylation product), the decarboxylation activity of SC-6 is increased by about 45 times, and with styrene as a substrate (cinnamic acid as a carboxylation product), its carboxylation activity is increased by nearly 20 times. The engineering strain SC-6 with efficient supply of cofactor prFMN has improved decarboxylation and carboxylation activity of Fdc, especially in the synthesis of high-value products such as cinnamic acid type, which has a wide range of application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering technology and fermentation engineering technology, and particularly relates to an engineering bacterium capable of efficiently supplying a cofactor prFMN of ferulic acid decarboxylase, and a construction method and application thereof. Background Art

[0002] Non-oxidative and reversible decarboxylases are important biocatalysts that can catalyze the decarboxylation of carboxylic acids and the reversible carboxylation of some products. The conditions are mild and the byproducts are few due to their high regioselectivity. However, chemical carboxylation reactions are usually carried out under high temperature and pressure, and the conditions are strict. Therefore, the use of decarboxylases to complete the carboxylation of compounds has attracted more and more attention. Among them, the ferulic acid decarboxylase Fdc (Ferulic acid decarboxylases) of the UbiD enzyme family from fungi such as Aspergillus niger, Saccharomyces cerevisiae and Candida dubliniensis is a typical representative, which depends on the cofactor isopropyl flavin mononucleotide prFMN (Prenylated flavin mononucleotide, prFMN). The above-mentioned fungal family members were originally found to act on lignin-derived ferulic acid. However, further studies have shown that they have a wide range of substrates characterized by α, β-unsaturated olefinic acids represented by cinnamic acid.

[0003] There are many methods for synthesizing cinnamic acid, mainly including various chemical methods such as Perkin reaction, benzaldehyde-acetone reaction, cinnamaldehyde oxidation reaction and styrene-carbon tetrachloride reaction. Most of them have the disadvantages of serious pollution, high energy consumption, low yield or high cost. Studies have found that the bioenzymatic method using styrene as a substrate - ferulic acid decarboxylase Fdc carboxylation reaction has the characteristics of mild conditions and environmental friendliness. Currently reported aromatic carboxyl acid decarboxylases including Fdc have problems such as low catalytic efficiency in the carboxylation direction. One of the main factors limiting the activity of Fdc is the poor stability and insufficient supply of its cofactor prFMN.

[0004] prFMN is a flavin cofactor required for the activity of isopropyl reversible decarboxylase discovered in recent years, and the UbiX family can utilize the reduced flavin FMN reduced Catalyzes the isopropyl transfer reaction of prenol and dimethylallyl (pyro)phosphate (DMAP(P)), forming a fourth non-aromatic ring by prenylation at the N5 and C6 positions to generate reduced prFMN reduced . Reduced prFMN reduced It is a product of UbiX catalysis and easily produces a series of oxidized substances after exposure to air.reduced After exposure to oxygen, the stable purple product prFMN was easily detected in both aqueous solution and in the UbiX-product complex. radical The active form of prFMN, prFMN, can only be generated in the presence of UbiD family enzymes such as Fdc. iminium , which is the only active form. And under continuous exposure to light, prFMN iminium prFMN ketamine Therefore, for Fdc, the series of enzymes and conditional control of the synthesis pathway of the active form of prFMN are crucial.

[0005] The study found that in the UbiD-UbiX E. coli co-expression system, due to the oxidized FMN oxidized The accumulation of UbiX makes it impossible to directly use the reduced FMN reduced , leading to the subsequent cofactor prFMN iminium Insufficient synthesis reduces the catalytic efficiency of Fdc. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an engineering bacterium and a construction method and application thereof for efficiently supplying the cofactor prFMN of ferulic acid decarboxylase, thereby improving the Fdc activity to efficiently synthesize cinnamic acid using styrene as a substrate.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] Ferulic acid decarboxylase Fdc is a prFMN-dependent reversible aromatic carboxylase from Saccharomyces cerevisiae, which is catalyzed by UbiX using reduced FMN. reduced It is produced by catalyzing the isopropyl transfer reaction of prenol and dimethylallyl (pyro)phosphate (DMAP(P)). In the original UbiX-UbiD system, two kinases, dimethylaminopyridine (DMAP) activating enzyme ThiM and riboflavin kinase RFK, are introduced to phosphorylate prenol to generate dimethylaminopyridine DMAP; riboflavin generates flavin mononucleotide FMN, generally oxidized FMN oxidized , UbiX cannot be used directly. On this basis, two reductases were introduced, flavin reductase Fre and formate dehydrogenase FDH. The former catalyzes the oxidation of FMN oxidized Generate reduced FMN reduced , reducing the oxidized FMN oxidizedThe accumulation of prFMN leads to insufficient supply of cofactor prFMN; the latter can continuously provide the required reducing power for the process, reducing coenzyme I nicotinamide adenine dinucleotide NADH (Nicotinamide adenine dinucleotide). Then the original UbiX-UbiD co-expression system was extended to the UbiX-Fdc-ThiM-RFK-Fre-FDH co-expression system, strengthening the synthesis of cofactor prFMN, which can solve the problem of insufficient supply of prFMN and improve the catalytic efficiency of Fdc.

[0009] The reaction principle of the present invention (such as Figure 1 As shown) as follows:

[0010] UbiX-UbiD starts with DMAP and reduced FMN reduced The cofactor prFMN is generated for the substrate to form the UbiX-prFMN complex, and then the cofactor prFMN is transferred to Fdc to form the Fdc-prFMN complex, which activates Fdc and catalyzes the carboxylation of styrene to produce cinnamic acid. reduced The precursor supply was used to construct ThiM to catalyze the production of DMAP from isoprenol as a substrate; RFK was constructed to catalyze riboflavin to produce oxidized FMN. oxidized , further construct Fre, using NADH as reducing force to catalyze FMN oxidized Restore to generate FMN reduced At the same time, FDH was constructed to consume NAD using formate as substrate + , thereby regenerating NADH.

[0011] The present invention provides a group of recombinant plasmids, including plasmid A, plasmid B and plasmid C, wherein the plasmid A includes the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the plasmid B includes the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, and the plasmid C includes the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] Furthermore, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.1 is the gene fdc of ferulic acid decarboxylase, and the nucleotide sequence shown in SEQ ID NO.2 is the gene ubix of isopropyltransferase.

[0013] Furthermore, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.3 is the gene thim of DMAP activating enzyme, and the nucleotide sequence shown in SEQ ID NO.4 is the gene rfk of riboflavin kinase.

[0014] Furthermore, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.5 is the flavin reductase gene fre, and the nucleotide sequence shown in SEQ ID NO.6 is the formate dehydrogenase gene fdh.

[0015] Furthermore, in the above technical solution, the vector of the recombinant plasmid includes one or more of pACYCDuet, pRSFDuet, and pETDuet.

[0016] The invention also provides a recombinant engineering bacterium expressing the group of recombinant plasmids.

[0017] Furthermore, in the above technical solution, the recombinant engineered bacteria is an engineered Escherichia coli bacteria.

[0018] Six enzymes are simultaneously overexpressed in the recombinant engineering bacteria, including flavin reductase Fre, formate dehydrogenase FDH, dimethylaminopyridine (DMAP) activating enzyme ThiM (Hydroxyethylthiazole kinase), riboflavin kinase RFK (Riboflavin kinase), isopropyltransferase UbiX and ferulic acid decarboxylase Fdc. The bacterial cells obtained by culturing the recombinant engineering bacteria are ultrasonically broken and the enzymes are released to obtain a whole-cell catalyst, which is added to a reaction system containing styrene to perform a catalytic reaction to synthesize cinnamic acid; or added to a reaction system containing cinnamic acid to perform a decarboxylation reaction to synthesize styrene.

[0019] The flavin reductase and DMAP activating enzyme are derived from the flavin reductase gene fre and the DMAP activating enzyme gene thim of Escherichia coli respectively; the formate dehydrogenase gene is derived from the formate dehydrogenase gene fdh of Pseudomonas sp. 101; the riboflavin kinase is derived from the rfk domain of the riboflavin kinase gene of Corynebacterium ammoniagenes; the isopropyl transferase is derived from the isopropyl transferase gene ubix of Pseudomonas aeruginosa; and the ferulic acid decarboxylase is derived from the ferulic acid decarboxylase gene fdc of Saccharomyces cerevisiae.

[0020] The present invention also provides a method for constructing the recombinant engineered bacteria, comprising the following steps:

[0021] 1) Clone the flavin reductase gene fre and formate dehydrogenase gene fdh, and connect the resulting genes to plasmid pETDuet by restriction endonuclease to obtain recombinant plasmid pETDuet_Fre_FDH; clone the DMAP activating enzyme gene ThiM and riboflavin kinase domain rfk, and connect the resulting genes to plasmid pRSFDuet by restriction endonuclease to obtain recombinant plasmid pRSFDuet_ThiM_RFK; clone the isopropyltransferase gene ubix and ferulic acid decarboxylase gene fdc, and connect the resulting genes to plasmid pACYCDuet by restriction endonuclease to obtain recombinant plasmid pACYCDuet_UbiX_Fdc.

[0022] 2) The three plasmids obtained in step 1) were introduced into competent E. coli BL21 (DE3) cells in equal amounts, and positive strains were screened based on resistance to three antibiotics: kanamycin, ampicillin and chloramphenicol to obtain recombinant engineered bacteria.

[0023] Furthermore, in the above technical solution, the resistance screening in step 2) is to spread the recombinant engineered bacteria on LB solid culture medium containing 50 μg / mL of kanamycin, ampicillin and chloramphenicol, and culture them overnight at 37°C until a single colony grows, and the obtained single colony is the positive strain.

[0024] The invention also provides the application of the recombinant engineering bacteria, which is applied to catalyzing styrene to synthesize cinnamic acid, and catalyzing cinnamic acid to decarboxylate to generate styrene.

[0025] Furthermore, in the above technical scheme, the conditions for catalyzing styrene to synthesize cinnamic acid include: using whole cells of recombinant engineered bacteria as catalysts, potassium bicarbonate as carbon source and buffer, and catalyzing styrene carboxylation to synthesize cinnamic acid at pH 8.1-8.3, normal temperature and normal pressure.

[0026] Furthermore, in the above technical solution, catalyzing the decarboxylation of cinnamic acid to produce styrene includes: using whole cells of recombinant engineered bacteria as catalysts and phosphate as a buffer, catalyzing the decarboxylation of cinnamic acid to produce styrene at a pH of 6.0-6.5, normal temperature and pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the enzyme reaction pathway related to the synthesis of ferulic acid decarboxylase Fdc and its cofactor prFMN.

[0028] Figure 2 A is a schematic diagram of the co-expression vector pETDuet-Fre-FDH of flavin reductase Fre and formate dehydrogenase FDH; Figure 2 B is a schematic diagram of the DMAP activating enzyme ThiM and riboflavin kinase RFK co-expression vector pRSFDuet-ThiM-RFK; Figure 2 C is a schematic diagram of the co-expression vector pACYCDuet-UbiX-Fdc of isopropyltransferase UbiX and ferulic acid decarboxylase Fdc.

[0029] Figure 3 The figure is the liquid phase detection diagram of cinnamic acid and styrene standard products. Among them, A is the liquid phase separation spectrum of cinnamic acid and styrene standard products, and B is the standard curve of cinnamic acid determined by liquid phase.

[0030] Figure 4 The decarboxylation activity of cinnamic acid in engineered bacteria was tested. A is the conversion rate of 5 mM cinnamic acid decarboxylation in 15 minutes for engineered bacteria transformed with different plasmids, and B is the Michaelis constant K of the kinetic parameters of SC-2 and SC-6. m and the maximum reaction rate V max .

[0031] Figure 5 This is a test of the styrene carboxylation activity of engineered bacteria. A is the amount of cinnamic acid produced by engineered bacteria transformed with different plasmids after overnight reaction with the same concentration of styrene for 24 hours, and B is the amount of cinnamic acid produced by SC-6 at different time points. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1: Construction of E. coli co-expression system

[0034] 1) The gene fdc (SEQ ID NO.1) encoding ferulic acid decarboxylase from Saccharomyces cerevisiae and the gene ubix (SEQ ID NO.2) encoding isopropyltransferase from Pseudomonas aeruginosa were cloned into the BamⅠ / HindⅢ restriction site and NdeⅠ / XhoⅠ restriction site of the expression vector pACYCDuet, respectively, to obtain the expression vector pACYCDuet-UbiX-Fdc (SEQ ID NO.7); the gene thim (SEQ ID NO.3) encoding DMAP activating enzyme from Escherichia coli and the gene rfk (SEQ ID NO.4) encoding riboflavin kinase from Corynebacterium ammoniagenes were cloned into the BamⅠ / HindⅢ restriction site and NdeⅠ / XhoⅠ restriction site of the same expression vector pRSFDuet, to obtain the expression vector pRSFDuet-ThiM-RFK (SEQ ID NO.8); the gene fre (SEQ ID NO.4) encoding flavin reductase from Escherichia coli was cloned into the BamⅠ / HindⅢ restriction site and NdeⅠ / XhoⅠ restriction site of the same expression vector pRSFDuet, to obtain the expression vector pRSFDuet-ThiM-RFK (SEQ ID NO.9); the gene fre (SEQ ID NO.5) encoding flavin reductase from Escherichia coli was cloned into the BamⅠ / HindⅢ restriction site and NdeⅠ / XhoⅠ restriction site of the same expression vector pRSFDuet, to obtain the expression vector pRSFDuet- NO.5) and the gene fdh (SEQ ID NO.6) encoding formate dehydrogenase from Pseudomonas were cloned into the BamⅠ / HindⅢ restriction site and NdeⅠ / XhoⅠ restriction site of the same expression vector pETDuet to obtain the expression vector pETDuet-Fre-FDH (SEQ ID NO.9), the map is shown in Figure 2 as shown in .

[0035] 2) The three expression vectors pACYCDuet-UbiX-Fdc, pRSFDuet-ThiM-RFK and pETDuet-Fre-FDH constructed in step 1) were all transformed into Escherichia coli competent cells BL21 (DE3), and the recombinant strains were obtained by screening on LB resistance plates containing 50 μg / mL kanamycin, ampicillin and chloramphenicol, and the recombinant strains were named SC-6; the expression vectors pACYCDuet-UbiX-Fdc and pRSFDuet-ThiM-RFK constructed in step 1) were transformed into Escherichia coli competent cells, and the recombinant strains were obtained by screening on LB resistance plates containing 50 μg / mL chloramphenicol and kanamycin, and the recombinant strains were named SC-4; the expression vector pACYCDuet-UbiX-Fdc constructed in step 1) was transformed into Escherichia coli competent cells, and the recombinant strains were obtained by screening on LB resistance plates containing 50 μg / mL chloramphenicol, and the recombinant strains were named SC-2.

[0036] Example 2: Cultivation and expression of recombinant E. coli

[0037] Select the recombinant engineering bacteria SC-6, SC-4 and SC-2 single colonies for strain culture, induced expression and purification verification. Inoculate the monoclonal positive strain into 5mL seed culture medium LB and culture it at 37℃ and 220rpm for 16-18 hours; then expand the seed liquid according to the inoculation volume ratio of 1:100, and finally inoculate it into 1L fermentation medium LB. First culture at 37℃ and 220rpm for 2-3 hours, and wait until OD 600 When the temperature reaches 0.6-0.8, adjust the temperature to 16°C. Add 0.2mM isopropylthiogalactoside IPTG, 1.0mM isoprenol, 0.1mM riboflavin and 1.0mM sodium formate to SC-6; add 0.2mM isopropylthiogalactoside IPTG, 1.0mM isoprenol and 0.1mM riboflavin to SC-4; add 0.2mM isopropylthiogalactoside IPTG to SC-2, and continue culturing for 12-16 hours.

[0038] The seed culture medium and fermentation culture medium LB formula are conventional LB formulas.

[0039] Example 3: Preparation of whole cell catalyst

[0040] The fermentation broth obtained by expression culture in Example 2 was centrifuged to collect the bacteria. After centrifugation at 5400 rpm and 4°C for 10 minutes for each 50 mL of bacterial solution, the supernatant was discarded, and then 5 mL of buffer (50 mM NaPi, pH 7.5; 50 mM NaCl; 1% glycerol) was added and the bacteria were resuspended by shaking. The resuspended bacterial solution was ultrasonically disrupted (300 W, 2 s working, 4 s rest) for 2 minutes to obtain the whole-cell catalyst.

[0041] Example 4: Cinnamic acid decarboxylation activity test of whole cell catalyst

[0042] Fdc decarboxylation activity test was performed using cinnamic acid as a substrate: in a 200 μL reaction system, 40 μL of the whole-cell catalyst suspension obtained in Example 3, approximately 2 mg of wet bacteria, 5 mM cinnamic acid, and 50 mM phosphate (pH 6.0) were added, and the reaction was carried out at 30°C for 15 minutes. When the reaction was completed, 600 μL of acetonitrile was added, and after shaking, the mixture was centrifuged at 12000 rpm and 4°C for 5 minutes, and 600 μL of the supernatant was taken into a brown liquid chromatography bottle. After testing, it was found that in the presence of 5 mM cinnamic acid at 15 minutes, the conversion rates of SC-4 and SC-6 were increased by about 25 times and 35 times, respectively, compared with SC-2. Among them, the conversion rate of SC-6 has reached 65%, and the decarboxylation activity has been greatly improved (see Figure 4 A).

[0043] Michaelis constant K m Maximum reaction rate V maxDetermination: In a 200 μL reaction system, 20 μL of the whole cell catalyst suspension obtained in Example 3, about 1 mg of bacteria, different concentrations of cinnamic acid (0.025, 0.05, 0.075, 0.10, 0.15, 0.20, 0.30, 0.60, 0.90, 1.20, 1.50 and 2.00 mM), 50 mM phosphate buffer (pH 6.0) were added, and the initial rate of the enzyme was determined at 30°C for 5 minutes. The basic relationship between substrate concentration and initial enzyme activity was substituted into the Michaelis-Menten function model to fit the Michaelis-Menten curve function expression of SC-2 and SC-6, and the corresponding maximum reaction rate V was calculated. max and Michaelis constant K m .

[0044] The maximum reaction rates of SC-2 and SC-6 were 0.17 + 0.01 μmoles / min / mg and 3.93 + 0.17 μmoles / min / mg, respectively, and the Michaelis constants were 91.10 + 3.63 μM and 232.78 + 18.15 μM, it can be seen that compared with SC-2, the Michaelis constant of SC-6 did not change significantly, while the maximum reaction rate increased by nearly 20 times (see Figure 4 B). Combined with the data of cinnamic acid conversion rate, it shows that the problem of insufficient supply of Fdc cofactor prFMN has been significantly improved.

[0045] Example 5: Activity test of styrene carboxylation to synthesize cinnamic acid using whole cell catalyst

[0046] Fdc carboxylation activity test was performed using styrene as a substrate: 90 μL of the whole cell catalyst suspension obtained in Example 3, about 3 mg of bacteria, 10 mM styrene, 1 M potassium bicarbonate were added to a 200 μL reaction system, the pH value of the solution was between 8.1 and 8.3, and the reaction was carried out at 30°C overnight. When the reaction was completed, 600 μL of acetonitrile was added, and the mixture was centrifuged at 12000 rpm and 4°C for 5 minutes after shaking. 600 μL of the supernatant was taken to a brown liquid chromatography bottle to detect the content of the generated cinnamic acid.

[0047] After overnight reaction for 24 hours, under the condition of 3 mg wet cells, the carboxylation activity of SC-6 increased from 0.7 μM to 15 μM of newly generated cinnamic acid compared with SC-2 (see Figure 5 A), the activity increased by nearly 20 times.

[0048] Determination of the enzymatic hydrolysis time point and its corresponding cinnamic acid concentration: In a 1 mL reaction system, 450 μL of the whole cell catalyst suspension obtained in Example 3, about 75 mg of bacteria (compared to the styrene carboxylation activity test, the enzyme amount was increased by 5 times), 10 mM styrene, 1 M potassium bicarbonate, the pH value of the solution was 8.1-8.3, and the reaction was carried out at a constant temperature of 30° C. After 4, 8, 12 and 24 hours, 200 μL of the reaction solution was taken out and terminated, and the generated cinnamic acid content was detected.

[0049] After testing, it was found that the production of cinnamic acid reached the maximum synthesis amount at 4 hours (see Figure 5 B).

[0050] The cinnamic acid content was determined by high performance liquid chromatography, and the specific detection conditions were: chromatographic column: Ecllpse XDB-C18 (250*4.6*5); mobile phase: acetonitrile: water = 1:1, 0.1% trifluoroacetic acid; detection wavelength: 254nm; injection volume: 5μL; column temperature: 25°C; flow rate: 1.0mL / min, isocratic elution for 20 minutes.

Claims

1. A recombinant engineered bacterium, characterized in that: The construction method includes the following steps: 1) Clone the flavin reductase gene fre and formate dehydrogenase fdh, and connect the resulting genes to the plasmid pETDuet by restriction endonucleases to obtain the recombinant plasmid pETDuet_Fre_FDH; clone the DMAP activating enzyme gene ThiM and riboflavin kinase domain rfk, and connect the resulting genes to the plasmid pRSFDuet by restriction endonucleases to obtain the recombinant plasmid pRSFDuet_ThiM_RFK; clone the isopropyltransferase gene ubix and ferulic acid decarboxylase gene fdc, and connect the resulting genes to the plasmid pACYCDuet by restriction endonucleases to obtain the recombinant plasmid pACYCDuet_UbiX_Fdc; 2) The three plasmids obtained in step 1) were introduced into competent E. coli BL21 (DE3) cells in equal amounts, and positive strains were screened for resistance to three antibiotics: kanamycin, ampicillin and chloramphenicol, to obtain recombinant engineered bacteria; The nucleotide sequence of the flavin reductase gene fre is shown in sequence SEQ ID NO.5, the nucleotide sequence of the formate dehydrogenase fdh is shown in sequence SEQ ID NO.6, the nucleotide sequence of the DMAP activating enzyme gene ThiM is shown in sequence SEQ ID NO.3, the nucleotide sequence of the riboflavin kinase gene rfk is shown in sequence SEQ ID NO.4, the nucleotide sequence of the ferulic acid decarboxylase gene fdc is shown in sequence SEQ ID NO.1, and the nucleotide sequence of the isopropyl transferase gene ubix is ​​shown in sequence SEQ ID NO.

2.

2. The use of the recombinant engineered bacteria according to claim 1, characterized in that: It is used to catalyze the synthesis of cinnamic acid from styrene and the decarboxylation of cinnamic acid to styrene.

3. The use according to claim 2, characterized in that: The conditions for catalyzing styrene to synthesize cinnamic acid include: using the whole cells of the recombinant engineering bacteria as a catalyst, potassium bicarbonate as a carbon source and a buffer, catalyzing styrene to synthesize cinnamic acid under the conditions of pH 8.1-8.3, normal temperature and normal pressure; The method of catalyzing the decarboxylation of cinnamic acid to generate styrene comprises: using the whole cell of the recombinant engineering bacteria as a catalyst and phosphate as a buffer, catalyzing the decarboxylation of cinnamic acid to generate styrene under the conditions of pH 6.0-6.5, normal temperature and normal pressure.

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