Method for producing p-coumaric acid through double-enzyme cascade catalysis

Abstract

The invention discloses a method for producing p-coumaric acid through double-enzyme cascade catalysis, which comprises the following steps: taking phenol, pyruvic acid and ammonium chloride as substrates, and carrying out cascade enzyme catalytic reaction on a tyrosine phenol lyase product and a tyrosine ammonia lyase product to synthesize p-coumaric acid. According to the method, phenol, pyruvic acid and ammonium chloride are used as substrates, a tyrosine phenol lyase product and a tyrosine ammonia lyase product are used as catalysts, the tyrosine phenol lyase product can convert the substrates into L-tyrosine, and the tyrosine ammonia lyase product can directly use the L-tyrosine as the substrate to convert the L-tyrosine into p-coumaric acid. Therefore, the tyrosine phenol lyase product and the tyrosine ammonia lyase product can realize cascade enzyme catalytic reaction in the same reactor, phenol with lower cost can be adopted as a conversion substrate, separation and purification of an intermediate product L-tyrosine are avoided, and the whole process flow is simple and efficient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the production of p-coumaric acid via a dual-enzyme cascade catalysis. Background Technology

[0002] p-Coumaric acid (4-hydroxycinnamic acid or trans-4-coumaric acid, p-CA), with the molecular formula C9H8O3, is an aromatic compound with broad application prospects. It serves as a precursor or intermediate for many compounds with high application value, and has important applications in pharmaceuticals, cosmetics, food, and chemical industries. For example, in the pharmaceutical field, p-coumaric acid can be used to develop and synthesize drugs with various biological activities such as antioxidation, anti-inflammation, antibacterial, and antitumor effects. In the cosmetics field, p-coumaric acid can be added to skincare products as a natural antioxidant to delay skin aging, reduce UV damage to the skin, and reduce skin glycation. In the food field, p-coumaric acid can be used as a flavoring agent and nutritional fortifier, as well as a natural preservative for food preservation. It can also be added to oils, meats, and other foods to prevent oxidative spoilage and extend shelf life.

[0003] Traditional p-coumaric acid production mainly relies on chemical synthesis, but this suffers from high production costs, low efficiency, and significant environmental pollution. Utilizing biosynthesis technology to produce p-coumaric acid offers advantages such as renewable raw materials, mild reaction conditions, and environmental friendliness, and can solve the problems of high raw material costs, complex processes, low efficiency, low product purity, and significant environmental pollution associated with existing p-coumaric acid production technologies. Bioconversion will become an important future development direction for p-coumaric acid production, driving its large-scale production and application.

[0004] For example, Chinese invention patent application CN113265431A discloses a method for synthesizing p-coumaric acid using p-hydroxyphenylpyruvic acid as a substrate and employing a multi-enzyme (carbonyl reductase, glucose dehydrogenase, and dehydratase) cascade catalysis. Although the production efficiency reaches 50 g / Lh, the cost of p-hydroxyphenylpyruvic acid substrate is high, and the p-coumaric acid product produced does not have a competitive advantage in the market.

[0005] For example, Chinese invention patent application CN117965514A discloses a method for preparing p-coumaric acid using L-tyrosine as a substrate and catalyzed by a mutant phenylalanine ammonia-lyase. The yield of p-coumaric acid using this method is only 3.12 g / L, and the substrate conversion rate is only 95%, making it difficult to achieve large-scale industrial production.

[0006] Currently, there is a huge demand for p-coumaric acid both domestically and internationally, and the market size is showing a steady growth trend. However, the production scale of p-coumaric acid is far from meeting the ever-increasing market demand. Therefore, it is urgent to develop an efficient p-coumaric acid production process to reduce production costs, promote large-scale industrial production of p-coumaric acid, and improve the market competitiveness of p-coumaric acid products. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the production of p-coumaric acid by a dual-enzyme cascade catalysis. This method has low production cost and high production efficiency, and can realize the large-scale industrial production of p-coumaric acid.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A method for the production of p-coumaric acid via a two-enzyme cascade catalysis, the method comprising: p-Coumaric acid was synthesized by using phenol, pyruvate, and ammonium chloride as substrates and by a cascade enzyme-catalyzed reaction with tyrosine phenol lyase and tyrosine ammonia-lyase products.

[0009] This invention uses phenol, pyruvate, and ammonium chloride as substrates, and tyrosine phenol lyase and tyrosine ammonia-lyase products as catalysts. The tyrosine phenol lyase product can convert the substrate into L-tyrosine, while the tyrosine ammonia-lyase product can directly use L-tyrosine as a substrate to convert L-tyrosine into p-coumaric acid. In this way, the tyrosine phenol lyase and tyrosine ammonia-lyase products can achieve a cascade enzyme catalytic reaction in the same reactor. This allows the use of low-cost phenol as a conversion substrate and avoids the separation and purification of the intermediate product L-tyrosine. The entire process is simple and efficient.

[0010] Preferably, in the tyrosine phenol lyase product, the amino acid sequence of the tyrosine phenol lyase is as shown in SEQ ID No. 1; The amino acid sequence of the tyrosine ammonia-lyase product is shown in SEQ ID No. 3.

[0011] When using the above-mentioned tyrosine phenol lyase and tyrosine ammonia-lyase products, not only is the substrate dosage high (substrate concentration can reach 5.2%), but the conversion rate is also high (the molar conversion rate of substrate is as high as 100% after 8 hours of conversion). The obtained p-coumaric acid conversion solution is free of phenol and L-tyrosine residues, which greatly facilitates the subsequent separation and purification of p-coumaric acid.

[0012] In this invention, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product can be added to the catalytic synthesis system in stages or sequentially, or they can be added to the catalytic synthesis system simultaneously.

[0013] When the tyrosine phenol lyase product and the tyrosine ammonia-lyase product are added sequentially to the catalytic synthesis system, the method for producing p-coumaric acid via a two-enzyme cascade catalysis includes the following two steps: S1. Using phenol, pyruvic acid, and ammonium chloride as substrates, and tyrosine phenol lyase product as catalyst, L-tyrosine is synthesized to obtain L-tyrosine conversion solution. S2. Using the L-tyrosine conversion solution as a substrate and the tyrosine ammonia-lyase product as a catalyst, p-coumaric acid is synthesized to obtain p-coumaric acid conversion solution.

[0014] Preferably, the tyrosine phenol lyase product is derived from the fermentation product of the tyrosine phenol lyase engineered bacteria, and is the fermentation cell, fermentation broth, or tyrosine phenol lyase extracted from the fermentation cell or fermentation broth of the tyrosine phenol lyase engineered bacteria. The tyrosine ammonia-lyase product is derived from the fermentation product of engineered tyrosine ammonia-lyase bacteria, and is the fermentation cell, fermentation broth, or tyrosine ammonia-lyase extracted from the fermentation cell or fermentation broth of engineered tyrosine ammonia-lyase bacteria.

[0015] Preferably, in step S1, the fermentation cells of engineered tyrosine phenol lyase are used as catalysts, and the catalytic synthesis system contains: 40-70 g / L phenol, 50-90 g / L sodium pyruvate, 20-60 g / L ammonium chloride, 0.05-0.2 g / L pyridoxal phosphate, and 10-40 g / L fermentation cells. The temperature of the catalytic synthesis system is 30-45℃ and the pH is 7.0-10.0.

[0016] Preferably, in step S2, the fermentation cells of engineered tyrosine ammonia-lyase bacteria are used as catalysts, the concentration of L-tyrosine in the catalytic synthesis system is 40-100 g / L, the concentration of fermentation cells is 10-40 g / L, the temperature of the catalytic synthesis system is 30-50℃, and the pH is 8.0-11.0.

[0017] When the tyrosine phenol lyase product and the tyrosine ammonia-lyase product are added simultaneously to the catalytic synthesis system, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product can be derived from different engineered bacteria, or they can be taken from the fermentation product of engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase. In this case, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product are a combination, which is the fermentation cell, fermentation broth of engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase, or the tyrosine phenol lyase and tyrosine ammonia-lyase extracted from the fermentation cell or fermentation broth.

[0018] Studies have found that, compared to adding them sequentially, when tyrosine phenol lyase and tyrosine ammonia-lyase are added simultaneously for one-step enzyme cascade catalysis, tyrosine phenol lyase converts the substrate to L-tyrosine, and tyrosine ammonia-lyase immediately converts L-tyrosine to p-coumaric acid. This not only shortens the conversion cycle by more than 50%, but also the ammonia produced during the conversion of L-tyrosine to p-coumaric acid can drive the equilibrium of tyrosine phenol lyase to shift towards the synthesis of L-tyrosine. This not only promotes the conversion efficiency of the substrate to L-tyrosine, but also achieves a higher yield of p-coumaric acid. When using fermentation products co-expressed with engineered bacteria, the fermentation cell cost can be further reduced by 50%.

[0019] Preferably, the fermentation method of the engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase is as follows: The seed culture of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase was inoculated into the fermentation medium. The fermentation system was controlled at a temperature of 32-42℃, a pH of 6.5-7.5, and dissolved oxygen above 30%. During fermentation, glycerol was continuously added at a rate of 100-150 mL / h to control the residual glycerol concentration in the fermentation system at 0.5-2 g / L and the cell growth rate at OD. 600 =3-4 / h; OD of bacterial cell concentration 600 When the temperature reaches 120°C or higher, the fermentation broth temperature is lowered and the cells are collected.

[0020] As a preferred embodiment, the method for producing p-coumaric acid by dual-enzyme cascade catalysis includes: using phenol, pyruvate, and ammonium chloride as substrates, and using fermentation cells of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase as catalysts to catalyze the synthesis of p-coumaric acid.

[0021] When fermentation cells are used directly as catalysts, not only is the catalytic efficiency high, but there is also no need to separate tyrosine phenol lyase and tyrosine ammonia-lyase, making the process simpler.

[0022] As a further preferred option, the catalytic synthesis system contains: 40-70 g / L phenol, 50-90 g / L sodium pyruvate, 20-60 g / L ammonium chloride, 0.05-0.2 g / L pyridoxal phosphate, 10-40 g / L tyrosine phenol lyase and tyrosine ammonia-lyase co-expressing fermentation cells of engineered bacteria; The temperature of the catalytic synthesis system is 30-45℃ and the pH is 7.0-10.0.

[0023] Compared with the prior art, the beneficial effects of the present invention are reflected in: (1) The present invention uses phenol, pyruvate and ammonium chloride as substrates and tyrosine phenol lyase product and tyrosine ammonia-lyase product as catalysts. The tyrosine phenol lyase product can convert the substrate into L-tyrosine, while the tyrosine ammonia-lyase product can directly use L-tyrosine as substrate to convert L-tyrosine into p-coumaric acid. In this way, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product can realize a cascade enzyme catalytic reaction in the same reactor. It can use phenol, which has a lower cost, as the conversion substrate and avoid the separation and purification of the intermediate product L-tyrosine. The whole process is simple and efficient.

[0024] (2) In this invention, tyrosine phenol lyase products and tyrosine ammonia-lyase products with specific amino acid sequences are used. These enzyme products can significantly increase the substrate dosage (the substrate concentration can reach 5.2%) and have a high conversion rate (the substrate molar conversion rate is as high as 100% after 8 hours of conversion). The obtained p-coumaric acid conversion solution has no phenol and L-tyrosine residues, which greatly facilitates the subsequent separation and purification of p-coumaric acid.

[0025] (3) In this invention, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product can be added to the catalytic synthesis system in stages or sequentially, or they can be added to the catalytic synthesis system simultaneously. However, studies have found that compared with adding them in stages, when the tyrosine phenol lyase product and the tyrosine ammonia-lyase product are added simultaneously for one-step enzyme cascade catalysis, the tyrosine phenol lyase can immediately convert L-tyrosine to p-coumaric acid after converting the substrate to L-tyrosine. This can not only shorten the conversion cycle by more than 50%, but also the ammonia produced during the conversion of L-tyrosine to p-coumaric acid can drive the equilibrium of the tyrosine phenol lyase to shift towards the synthesis of L-tyrosine. This can not only promote the conversion efficiency of the substrate to L-tyrosine, but also obtain a higher p-coumaric acid yield. When using fermentation products of co-expressed engineered bacteria, the fermentation cell cost can be further reduced by 50%. Attached Figure Description

[0026] Figure 1 pCDFDuet- is a recombinant expression vector for tyrosine phenol lyase. tpl Structural diagram; Figure 2 Electrophoresis diagrams showing the correct expression of the corresponding enzymes in engineered bacteria of tyrosine phenol lyase, engineered bacteria of tyrosine ammonia-lyase, and engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase. Wherein, TPL represents tyrosine phenol lyase, TAL represents tyrosine ammonia-lyase, M is a protein molecular marker, lanes 1 and 3 are negative controls, lane 2 is the engineered bacteria for tyrosine phenol lyase, lane 4 is the engineered bacteria for tyrosine ammonia-lyase, and lane 5 is the engineered bacteria for co-expression of tyrosine phenol lyase and tyrosine ammonia-lyase; the same applies below. Figure 3The cumulative cell count of the engineered tyrosine phenol lyase strain at various time points during fermentation was statistically analyzed. Figure 4 Statistics on product yield at each time point during the preparation of L-tyrosine using phenol as a substrate enzyme; Figure 5 pET24a- is a recombinant expression vector for tyrosine ammonia-lyase. tal Structural diagram; Figure 6 The cumulative cell count of engineered tyrosine ammonia-lysin-producing bacteria at various time points during fermentation was statistically analyzed. Figure 7 Statistics on substrate conversion rates at various time points when using a conversion solution containing L-tyrosine as a substrate enzyme to catalyze the preparation of p-coumaric acid; Figure 8 The liquid chromatogram of the p-coumaric acid product obtained in Example 1; Where min represents the retention time (minutes), the same applies below; Figure 9 To statistically analyze the product yield at various time points when using engineered bacteria expressing tyrosine phenol lyase and tyrosine ammonia-lyase in a one-step catalytic process to prepare p-coumaric acid from phenol substrate; Figure 10 The liquid chromatogram of the p-coumaric acid product obtained in Example 2; Figure 11 The cumulative cell count of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase at various time points during fermentation was statistically analyzed. Figure 12 To statistically analyze the product yield at various time points when preparing p-coumaric acid from phenol substrate using a one-step catalytic method catalyzed by co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase. Figure 13 This is the liquid chromatogram of the p-coumaric acid conversion solution in Example 3; Figure 14 The liquid chromatogram of the p-coumaric acid product obtained in Example 3; Figure 15 This is a synthetic route diagram for the production of p-coumaric acid by a dual-enzyme cascade catalysis method according to the present invention. Detailed Implementation

[0027] The following examples illustrate the technical solution of the present invention in a more detailed manner.

[0028] Example 1: Two-step enzymatic production of p-coumaric acid This embodiment describes a method for producing p-coumaric acid via a two-enzyme cascade catalysis, comprising the following steps: S1. Using phenol, pyruvic acid, and ammonium chloride as substrates, and the fermentation cells of engineered tyrosine phenol lyase bacteria as catalysts, L-tyrosine is synthesized to obtain L-tyrosine conversion solution. Specifically, including: (1) Construction of the recombinant expression vector pCDFDuet- for tyrosine phenol lyase tpl This embodiment screened and compared tyrosine phenol lyases from various microbial sources, and screened a phenol lyase TPL (amino acid sequence as shown in SEQ ID No. 1) with excellent enzymatic properties. This enzyme can efficiently catalyze the biosynthesis of L-tyrosine from substrates such as phenol. In order to increase the protein expression level of tyrosine phenol lyase in the *E. coli* expression host and improve enzyme activity, this embodiment optimized the codon sequence of tyrosine phenol lyase TPL according to the codon preference of *E. coli* expression. The optimized phenol lyase... tal The gene nucleic acid sequence was handed over to Qingke Biotechnology Co., Ltd. for whole-gene synthesis (nucleotide sequence as shown in SEQ ID No. 2), and then the synthesized... tal The gene fragment was cloned into the E. coli prokaryotic expression vector pCDFDuet-1 to obtain the tyrosine phenol lyase recombinant expression vector pCDFDuet- tpl .

[0029] The specific construction process is as follows: First, the pCDFDuet-1 expression vector is used with restriction endonucleases. Nco I and Not l Ⅰ Perform double enzyme digestion, separate the digestion products by nucleic acid electrophoresis, and then recover the DNA using a gel to obtain the linearized pCDFDuet-1 vector fragment; the fully synthesized plasmid PUC- containing the TPL gene fragment is then... tpl Using restriction endonucleases Nco I and Not I. The TPL gene fragment was subjected to double enzyme digestion, and the digestion products were separated by nucleic acid electrophoresis. The fragment was then recovered via DNA gel extraction to obtain the TPL gene fragment. The TPL gene fragment was then reacted with restriction endonuclease-treated DNA fragments. Nco I and Not l Ⅰ The linearized pCDFDuet-1 vector was cloned and recombined in one step using a gene cloning kit. The recombinant product was transformed into *E. coli* DH5α competent cells, and the transformation medium was plated on LB agar plates (containing 50 μg / mL streptomycin). Transformants grown on the plates were picked and transferred to 5 mL LB tubes (containing 50 μg / mL streptomycin) and cultured overnight at 37°C with a shaker for 16 h. The transformant plasmids were extracted from the transformant culture using a plasmid extraction kit and screened by nucleic acid electrophoresis to obtain the recombinant expression vector pCDFDuet-1. tpl (See Figure 1 ).

[0030] (2) Construction of engineered strains of tyrosine phenol lyase Specifically, the recombinant expression vector pCDFDuet- tplThe plasmid was added to *E. coli* BL21(DE3) competent cells and incubated on ice for 30 minutes; followed by heat shock at 42°C for 90 seconds and then on ice for 2 minutes; 1 ml of LB medium was added and the cells were incubated at 37°C for 1 hour; the transformation solution was plated on LB agar plates (with 50 mg / L streptomycin) and incubated at 37°C for 24 hours; single colonies that had grown well on the plates were inoculated into 10 mL LB shake flasks (1% peptone, 0.5% yeast extract, 1% sodium chloride, 50 mg / L streptomycin) and incubated at 37°C and 250 rpm for 12 hours. Then, 50 mL LB shake flasks were inoculated at a 10% inoculum size (50 mg / L streptomycin) and incubated at 37°C and 250 rpm for approximately 3 hours. When the OD600 of the culture medium reached 3-5, 0.2 mM IPTG was added to a final concentration, and the cells were cooled to 28°C and incubated overnight. After 12 hours of induction culture, 2 mL of culture medium was collected, centrifuged at 12000 rpm for 1 min, and the bacterial cells were collected. After removing the supernatant, 1 mL of water was added to suspend the bacterial cells, and the cells were sonicated for 2 min to disrupt the cells. 0.2 mL of the cell disruption solution was added to 0.04 mL of 5X protein loading buffer, and after boiling for 10 min, 5 μL of the sample was taken for SDS-PAGE protein electrophoresis. The target protein band was around 50 kDa, indicating that the tyrosine phenol lyase gene TPL had been successfully introduced into the E. coli host, and that the TPL protein was expressed in large quantities and correctly (see...). Figure 2 This indicates that the engineered tyrosine phenol lyase strain TPL / BL21(DE3) has the ability to biocatalyze the preparation of L-tyrosine using phenol as a substrate.

[0031] (3) High-density fermentation induction of tyrosine phenol lyase engineered strain TPL / BL21(DE3) Specifically, the engineered tyrosine phenol lyase strain TPL / BL21(DE3) was inoculated from frozen glycerol tubes into LB solid medium containing the corresponding antibiotics (the medium composition was: 1% peptone, 0.5% yeast extract, 1% sodium chloride, with 50 mg / L streptomycin added according to the resistance of the expression vector in the engineered strain) for streaking isolation, and incubated at 37℃ for 24 h until single colonies grew; single colonies were picked into test tubes containing 5 mL of LB liquid medium (50 mg / L streptomycin), and incubated at 220 rpm and 37℃ for 12-16 h to obtain primary seed culture; 1 mL of the cultured primary seed culture was inoculated into 500 mL of LB liquid medium (50 mg / L streptomycin), and incubated at 220 rpm and 37℃ for 12 h to obtain secondary seed culture.

[0032] 500 mL of the cultured secondary seed was inoculated at a 10% inoculation rate into 5 L of sterilized fermentation medium (5 L of medium was prepared for a 15 L fermenter; the medium composition was as follows: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K₂HPO₄, 2.2 g / L KH₂PO₄, 5 g / L glycerol; sterilized at 121℃ for 30 minutes, cooled to 37℃, and the pH was adjusted to 7.0 with 20% ammonia before inoculation). The fermentation was carried out at 37℃, 300 rpm, and an air flow rate of 300 L / h. During fermentation, the pH was maintained at 7.0 with 20% ammonia, and the rotation speed (300-600 rpm) and air flow rate (300-600 L / h) were adjusted sequentially to maintain dissolved oxygen above 30%. When the glycerol in the medium was depleted, the dissolved oxygen and pH rose sharply. At this point, 50% glycerol (sterilized at 121℃ for 30 minutes) was added at a feed rate of 100 mL / h. When the cell concentration (OD) in the fermentation broth 600 Once the concentration reaches 30 or higher, lower the temperature to 28℃ and add 1 mM IPTG inducer to induce protein expression. Samples were taken every 3 hours to measure the cell concentration and glycerol concentration in the fermentation broth. The residual glycerol concentration was controlled to be below 1 g / L by adjusting the feeding rate to 100-150 mL / h. The cell growth rate OD was also monitored. 600 =3-4 / h, until the cell concentration continuously increases during fermentation, and the cell concentration OD after 36 h of fermentation reaches 3-4 h. 600 The concentration of the bacterial culture should reach 120 or higher, and the wet cell count should reach 15% or higher. Cool the fermentation broth to 15°C, collect the cells by tubular centrifugation, and store at -20°C for later use (see [link to fermentation instructions]). Figure 3 ).

[0033] (4) Enzyme-catalyzed synthesis of L-tyrosine Specifically, add 4L of pure water, 1g of pyridoxal phosphate (PLP), 5g of ethylenediaminetetraacetic acid (EDTA), and 350g of ammonium chloride to a 10L conversion tank. Stir at 300 rpm and heat to 37°C. Adjust the pH to 8.2 with 40% NaOH. Add 200g of tyrosine phenol lyase cells. Add substrate (715g sodium pyruvate, 520g phenol, and pure water to 5L, stirring constantly) at a rate of 1L / h. Complete substrate addition in 5 hours, maintaining pH 8.2 with 40% NaOH throughout the process. Continue the reaction after the pH rises until the residual phenol substrate is zero according to liquid chromatography, approximately 7 hours later. Figure 4 It can be seen that the yield of L-tyrosine reached 100.2 g / L during the conversion process, and the conversion rate of phenol substrate reached 100%, indicating that all substrates were completely converted. The L-tyrosine conversion solution was heated to 80℃ and held for 30 minutes for inactivation treatment, and then cooled to 40℃ to serve as the substrate for the next step of catalytic preparation of p-coumaric acid.

[0034] S2. Using L-tyrosine conversion solution as substrate and tyrosine ammonia-lyase product as catalyst, p-coumaric acid is synthesized to obtain p-coumaric acid conversion solution. Specifically, it includes: (1) Constructing the recombinant expression vector pET24a- of tyrosine ammonia-lyase tal Tyrosine ammonia-lyase TAL can catalyze the biosynthesis of p-coumaric acid from L-tyrosine substrates. In this embodiment, through screening and comparison of tyrosine ammonia-lyases from various microbial sources, a tyrosine ammonia-lyase TAL with excellent enzymatic properties was selected. Subsequently, the catalytic active sites of wild-type tyrosine ammonia-lyase were analyzed using the protein analysis website https: / / consurf.tau.ac.il. A total of 5 catalytic active site sites (Y87, Q108, P239, Y340, P375) were predicted. Single-site directed mutations and multiple-site superimposed combination mutations were performed on these 5 catalytic active sites to obtain the following single mutants: Y87D, Y87T, Y87A, Q108M, Q108S, Q108G, P239R, P239Y, P239V, Y340Q, Y340P, Y340M, P375T, P375K, P375H and multiple mutants: Q108S / Y340Q, Y340Q / P375H, Q108S / P375H, Q108S / Y340Q / P375H.

[0035] Then, based on the codon preference of *E. coli*, the sequences of tyrosine ammonia-lyase TAL and its various mutants were codon optimized. The optimized tyrosine ammonia-lyase TAL and its mutant gene nucleic acid sequences were then sent to Qingke Biotechnology Co., Ltd. for whole-genome synthesis. The synthesized TAL gene fragment was then cloned into the *E. coli* prokaryotic expression vector pET24a, resulting in the recombinant expression vectors pET24a- for each tyrosine ammonia-lyase. tal .

[0036] The specific construction process is as follows: First, the pET-24a(+) expression vector is double-digested with restriction endonucleases BamH Ⅰ and Xhol Ⅰ. The digestion products are separated by nucleic acid electrophoresis, and then the DNA is recovered by gel extraction to obtain the linearized pET-24a(+) vector fragment; the fully synthesized plasmid PUC- containing the TAL gene fragment is then... talThe DNA was double-digested with restriction endonucleases BamH I and Xhol I. The digestion products were separated by nucleic acid electrophoresis, and the gene fragment was then recovered by DNA gel extraction to obtain the TAL gene fragment. The TAL gene fragment was then cloning and recombination with the pET-24a(+) vector linearized with restriction endonucleases BamH I and Xhol I using a gene cloning kit. The recombinant product was transformed into *E. coli* DH5α competent cells, and the transformation solution was plated on LB agar plates (with 50 μg / mL kanamycin). Transformants grown on the plates were picked and transferred to 5 mL LB tubes (with 50 μg / mL kanamycin) and incubated overnight at 37°C with a shaker for 16 h. Transformant plasmids were extracted from the transformant culture using a plasmid extraction kit and screened by nucleic acid electrophoresis to obtain the recombinant expression vector. pET24a-tal (See Figure 5 ).

[0037] (2) Construction of engineered tyrosine ammonia-lysin-producing bacteria TAL / BL21(DE3) Specifically, the recombinant expression vector pET24a- tal The plasmid was added to *E. coli* BL21(DE3) competent cells and incubated on ice for 30 minutes; followed by heat shock at 42°C for 90 seconds and then on ice for 2 minutes; 1 ml of LB medium was added and the cells were incubated at 37°C for 1 hour; the transformation solution was plated on LB agar plates (containing 50 mg / L kanamycin) and incubated at 37°C for 24 hours; single colonies that had grown well on the plates were inoculated into 10 mL LB shake flasks (1% peptone, 0.5% yeast extract, 1% sodium chloride, 50 mg / L kanamycin) and incubated at 37°C and 250 rpm for 12 hours. Then, 50 mL LB shake flasks (50 mg / L kanamycin) were inoculated at a 10% inoculum and incubated at 37°C and 250 rpm for approximately 3 hours. When the OD600 of the culture medium reached 3-5, 0.2 mM IPTG was added to a final concentration, and the cells were cooled to 28°C and incubated overnight. After 12 hours of induction culture, 2 mL of culture medium was collected, centrifuged at 12000 rpm for 1 min, and the bacterial cells were collected. After removing the supernatant, 1 mL of water was added to suspend the bacterial cells, and the cells were sonicated for 2 min to disrupt the cells. 0.2 mL of the cell disruption solution was added to 0.04 mL of 5X protein loading buffer, and after boiling for 10 min, 5 μL of the sample was taken for SDS-PAGE protein electrophoresis. The target protein band was around 70 kDa, indicating that the tyrosine ammonia-lyase gene TAL had been successfully introduced into the E. coli host, and that the TAL protein was expressed in large quantities and correctly (see...). Figure 2 In this embodiment, the engineered tyrosine ammonia-lysin TAL / BL21(DE3) strains have the ability to prepare p-coumaric acid by biocatalysis using L-tyrosine as a substrate.

[0038] After inducing expression in various engineered bacteria of tyrosine ammonia-lyase (including wild type and various mutants), bacterial cells were collected, and the enzyme activities of wild-type and mutant tyrosine ammonia-lyase were analyzed. The analytical method was as follows: 10g of L-tyrosine substrate was weighed into a 500mL shake flask, 80g of pure water was added, the pH was adjusted to 10 with 40% NaOH, 2g of bacterial enzyme was added, and the reaction was carried out at 40℃ and 220r / min for 1h in a shaker. The concentration of p-coumaric acid in the transformation solution was detected by liquid chromatography. The specific activity of bacterial enzyme was determined according to the amount of p-coumaric acid generated. The relative activity values ​​of mutant enzymes were calculated and compared (with the wild-type tyrosine ammonia-lyase activity as 100%). The results are shown in Table 1.

[0039] Enzyme activity definition (U / g): The amount of enzyme consumed to produce 1 μmol of p-coumaric acid per minute.

[0040] The detection method for coumaric acid is as follows: C18 column, mobile phase 1‰ acetic acid / methanol = 55 / 45, flow rate 0.6 mL / min, column temperature 30℃, detection wavelength 280 nm, sample diluted 1000 times with methanol before injection into liquid chromatography, retention time 25 minutes.

[0041] Table 1 Comparison of catalytic abilities of different tyrosine ammonia-lysinases As shown in Table 1, the enzyme activities of tyrosine ammonia-lyase mutants varied significantly depending on the mutation site and the mutation target, even when the mutation site was the same. Specifically, single mutations of Q108S, Y340Q, and P375H increased the enzyme activity of the tyrosine ammonia-lyase mutant by 56%, 37%, and 49%, respectively. When combining mutations of two or more sites at single mutation sites that enhanced enzyme activity, double mutations did not always result in an additive increase in enzyme activity. Compared to single mutations, only the Q108S / Y340Q and Q108S / P375H double mutations produced a synergistic effect. However, the Q108S / Y340Q / P375H triple mutation increased the enzyme activity of the tyrosine ammonia-lyase mutant by 102%.

[0042] Therefore, this embodiment uses a mutant tyrosine ammonia-lyase TAL. Q108S / Y340Q / P375H (Amino acid sequence as shown in SEQ ID No. 3, corresponding nucleotide sequence as shown in SEQ ID No. 4) The engineered bacteria TAL / BL21(DE3) is used as a catalyst to proceed to the next step.

[0043] (3) High-density fermentation induction of tyrosine ammonia-lyase engineered strain TAL / BL21(DE3) The high-density fermentation induction method for the tyrosine ammonia-lyase engineered bacterium TAL / BL21(DE3) is the same as that for the tyrosine phenol lyase engineered bacterium TPL / BL21(DE3). The cumulative cell count of the tyrosine ammonia-lyase engineered bacterium at various time points during fermentation is shown in the figure below. Figure 6 As shown.

[0044] (4) Catalytic synthesis of p-coumaric acid Specifically, 5 L of L-tyrosine conversion solution was added to a 10 L conversion tank, stirred at 300 rpm, and heated to 40°C. The pH was adjusted to 10 with 40% NaOH. 10 g of sodium bisulfite was added and stirred thoroughly. 200 g of engineered tyrosine ammonia-lyase bacteria TAL / BL21(DE3) cells were added. Nitrogen gas was introduced through the air line to maintain the tank pressure at 0.05 MPa to begin the conversion. During the conversion, the pH was maintained at 10 with 40% NaOH. The p-coumaric acid content and substrate residue in the p-coumaric acid conversion solution were monitored during the process until the substrate reduction was slow. The conversion was completed in 10 h, with a substrate molar conversion rate of 99.6% (see...). Figure 7 ).

[0045] (5) Extract pure p-coumaric acid from p-coumaric acid conversion solution Specifically, take 1 L of p-coumaric acid conversion solution, add 3% diatomaceous earth and stir at 40℃ for 0.5 h. Filter the solution using a Buchner funnel to separate the solid and liquid components. Add 0.3% activated carbon to the p-coumaric acid filtrate, stir at 40℃ for 1 h and then filter. Adjust the pH of the filtrate to 2-3 with 30% sulfuric acid. Cool the acid-adjusting solution to 15-20℃ and maintain for 2 h. Filter the crystallization solution to obtain wet crude p-coumaric acid. Wash the wet crude product three times with 1 volume of pure water. Dry the obtained wet product in an oven at 80℃ to constant weight to obtain pure p-coumaric acid.

[0046] In this embodiment, the mass conversion yield of p-coumaric acid catalyzed by tyrosine ammonia-lyase was over 85%, and the extraction yield of p-coumaric acid during the entire extraction process could reach over 96%. The obtained p-coumaric acid product contained 99.10% p-coumaric acid, 0.76% moisture, 0.05% residue, and a liquid chromatography purity of 99.67%. No phenol or L-tyrosine substrate residues were detected in the product, and the product quality was qualified. Figure 8 ).

[0047] Example 2 This embodiment discloses a method for the production of p-coumaric acid via a dual-enzyme cascade catalysis, comprising: using phenol, pyruvate, and ammonium chloride as substrates, and using fermentation cells of engineered tyrosine phenol lyase and engineered tyrosine ammonia-lyase as catalysts to catalyze the synthesis of p-coumaric acid.

[0048] Specifically, it includes the following steps: (1) Construction of the recombinant expression vector pCDFDuet- tpl and pET24a- tal The corresponding part is the same as in Example 1.

[0049] (2) Constructing engineered strains of tyrosine phenol lyase and tyrosine ammonia-lyase The corresponding part is the same as in Example 1.

[0050] (3) High-density fermentation induction of engineered tyrosine phenol lyase and engineered tyrosine ammonia-lyase bacteria The corresponding part is the same as in Example 1.

[0051] (4) Catalytic synthesis of p-coumaric acid Specifically, add 4L of pure water, 10g of sodium bisulfite, 1g of pyridoxal phosphate (PLP), 5g of ethylenediaminetetraacetic acid (EDTA), and 350g of ammonium chloride to the conversion tank. Stir at 300 rpm, heat to 37°C, adjust the pH to 9.0 with 40% NaOH, and add 200g of engineered tyrosine phenol lyase cells and 200g of engineered tyrosine ammonia-lyase cells. Introduce nitrogen through the air line to maintain a tank pressure of 0.05 MPa and begin conversion. Add substrate (715g of sodium pyruvate, 520g of phenol, and pure water to 5L, stirring constantly) at a rate of 1L / h. Complete substrate addition after 5 hours. Maintain the pH at 9.0 with 40% NaOH throughout the process. Monitor the p-coumaric acid content in the p-coumaric acid conversion solution, as well as the phenol substrate and L-tyrosine intermediate content. Continue conversion until the residue is zero, approximately 9 hours later. (See [link to relevant documentation]) Figure 9 The phenol substrate conversion rate reached 100%, and all substrates were completely converted.

[0052] (5) Extract pure p-coumaric acid from p-coumaric acid conversion solution The corresponding part is the same as in Example 1.

[0053] In this embodiment, the mass conversion yield of p-coumaric acid catalyzed by tyrosine ammonia-lyase was over 88%, and the extraction yield of p-coumaric acid during the entire extraction process could reach over 96%. The obtained p-coumaric acid product contained 99.30% p-coumaric acid, 0.51% moisture, 0.04% residue, and a high purity of 99.62% according to liquid chromatography. No phenol or L-tyrosine substrate residues were detected in the product, and the product quality was qualified (see...). Figure 10 ).

[0054] Example 3 This embodiment discloses a method for the production of p-coumaric acid via a dual-enzyme cascade catalysis, comprising: using phenol, pyruvate, and ammonium chloride as substrates, and using fermentation cells of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase as catalysts to catalyze the synthesis of p-coumaric acid.

[0055] Specifically, it includes the following steps: (1) Construction of the recombinant expression vector pCDFDuet- tpl and pET24a- tal The corresponding part is the same as in Example 1.

[0056] (2) Constructing engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase The specific construction process is as follows: The recombinant expression vector pCDFDuet- tpl and pET24a- tal The plasmid was added to *E. coli* BL21(DE3) competent cells and incubated on ice for 30 minutes; followed by heat shock at 42°C for 90 seconds and then on ice for 2 minutes; 1 ml of LB medium was added and the cells were incubated at 37°C for 1 hour; the transformation solution was plated on LB agar plates (with 50 mg / L streptomycin and 50 mg / L kanamycin) and incubated at 37°C for 24 hours; single colonies that had grown well on the plates were inoculated into 10 mL LB shake flasks (1% peptone, 0.5% yeast extract, 1% sodium chloride, with 50 mg / L streptomycin and 50 mg / L kanamycin) and incubated at 37°C and 250 rpm for 12 hours. A 10% inoculum was then inoculated into 50 mL LB shake flasks (with 50 mg / L streptomycin and 50 mg / L kanamycin) and incubated at 37°C and 250 rpm for approximately 3 hours. When the OD600 of the culture medium reached 3-5, 0.2 mM IPTG was added to a final concentration, and the cells were cooled to 28°C and incubated overnight. After 12 hours of induction culture, 2 mL of culture medium was collected and centrifuged at 12000 rpm for 1 min. The supernatant was removed, and 1 mL of water was added to suspend the cells. The cells were then sonicated for 2 min to disrupt the cell lysis. 0.2 mL of the cell lysis buffer was added to 0.04 mL of 5X protein loading buffer, and the mixture was incubated in a boiling water bath for 10 min. 5 μL of the sample was then subjected to SDS-PAGE protein electrophoresis. The gel image showed TPL (50 kDa) and TAL (70 kDa) protein bands of the same size as those observed during the previous recombinant enzyme expression. The protein electrophoresis confirmed the presence of the tyrosine phenol lyase gene. tpl The tyrosine ammonia-lyase gene tal has been successfully introduced into the E. coli host, and both TPL and TAL proteins are expressed in large quantities and correctly (see [link]). Figure 2 The engineered strain TPL-TAL / BL21(DE3), which co-expresses tyrosine phenol lyase and tyrosine ammonia-lyase, has been shown to have the ability to prepare p-coumaric acid using a two-enzyme cascade catalysis with phenol, pyruvate, and ammonium as substrates.

[0057] (3) High-density fermentation induction of engineered strain TPL-TAL / BL21(DE3) co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase The high-density fermentation induction method for the engineered strain TPL-TAL / BL21(DE3) co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase was the same as that for the engineered strain TPL / BL21(DE3) expressing tyrosine phenol lyase. The cumulative cell count of the engineered strain co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase at various time points during fermentation is shown in the figure below. Figure 11 As shown.

[0058] (4) Catalytic synthesis of p-coumaric acid Specifically, add 4L of pure water, 10g of sodium bisulfite, 1g of pyridoxal phosphate (PLP), 5g of ethylenediaminetetraacetic acid (EDTA), and 350g of ammonium chloride to the conversion tank. Stir at 300 rpm, raise the temperature to 37°C, adjust the pH to 9.0 with 40% NaOH, and add 200g of tyrosine phenol lyase and tyrosine ammonia-lyase co-expressing cells. Introduce nitrogen through the air line to maintain a tank pressure of 0.05 MPa to begin conversion. Add substrate (715g of sodium pyruvate, 520g of phenol, and pure water to 5L, stirring constantly) at a rate of 1L / h. Complete substrate addition in 5 hours. Maintain the pH at 9.0 with 40% NaOH throughout the process. Monitor the p-coumaric acid content in the p-coumaric acid conversion solution during the process (see...). Figure 12 Simultaneously, the content of phenol substrate and L-tyrosine intermediate was detected, and the conversion was completed in about 8 hours until the residue was 0 (see [link]). Figure 13 The phenol substrate conversion rate reached 100%, and all substrates were completely converted.

[0059] (5) Extract pure p-coumaric acid from p-coumaric acid conversion solution The corresponding part is the same as in Example 1.

[0060] In this embodiment, the mass conversion yield of p-coumaric acid catalyzed by tyrosine ammonia-lyase was over 90% (basically reaching the theoretical mass conversion rate). The extraction yield of p-coumaric acid during the entire extraction process reached over 96%. The obtained p-coumaric acid product contained 99.50% p-coumaric acid, 0.45% moisture, 0.03% residue, and had a liquid chromatography purity of 99.87%. No phenol or L-tyrosine substrate residues were detected in the product, indicating that the product quality was qualified (see...). Figure 14 ).

[0061] The synthetic route of this invention, which involves converting substrates such as phenol to L-tyrosine and then converting L-tyrosine to p-coumaric acid, is shown in the diagram below. Figure 15 As shown. From Figure 15As can be seen, when two enzymes (tyrosine phenol lyase and tyrosine ammonia-lyase), substrates (pyruvate, phenol, ammonia) and cofactors (PLP) are placed in the same reactor and reacted simultaneously, not only can the separation and purification of the intermediate product L-tyrosine be avoided, simplifying the process, but also the NH3 generated during the conversion of L-tyrosine to p-coumaric acid can drive the equilibrium of tyrosine phenol lyase to shift towards the synthesis of L-tyrosine. This is the main reason why the conversion cycle of Examples 2 and 3 is shorter (50% shorter) than that of Comparative Example 1 and the p-coumaric acid yield is higher than that of Comparative Example 1.

Claims

1. A method for the production of p-coumaric acid via a two-enzyme cascade catalysis, characterized in that, include: p-Coumaric acid was synthesized by using phenol, pyruvate, and ammonium chloride as substrates and by a cascade enzyme-catalyzed reaction with tyrosine phenol lyase and tyrosine ammonia-lyase products.

2. The method as described in claim 1, characterized in that, The amino acid sequence of the tyrosine phenol lyase product is shown in SEQ ID No. 1; The amino acid sequence of the tyrosine ammonia-lyase product is shown in SEQ ID No.

3.

3. The method as described in claim 1, characterized in that, The method involves sequentially adding the tyrosine phenol lyase product and the tyrosine ammonia-lysin product to a catalytic synthesis system, wherein the method comprises: S1. Using phenol, pyruvic acid, and ammonium chloride as substrates, and tyrosine phenol lyase product as catalyst, L-tyrosine is synthesized to obtain L-tyrosine conversion solution. S2. Using the L-tyrosine conversion solution as a substrate and the tyrosine ammonia-lyase product as a catalyst, p-coumaric acid is synthesized to obtain p-coumaric acid conversion solution.

4. The method as described in claim 3, characterized in that, The tyrosine phenol lyase product is derived from the fermentation product of engineered tyrosine phenol lyase bacteria, and is the fermentation cell, fermentation broth, or tyrosine phenol lyase extracted from the fermentation cell or fermentation broth of engineered tyrosine phenol lyase bacteria. The tyrosine ammonia-lyase product is derived from the fermentation product of engineered tyrosine ammonia-lyase bacteria, and is the fermentation cell, fermentation broth, or tyrosine ammonia-lyase extracted from the fermentation cell or fermentation broth of engineered tyrosine ammonia-lyase bacteria.

5. The method as described in claim 4, characterized in that, In step S1, the fermentation cells of engineered tyrosine phenol lyase are used as catalysts. The catalytic synthesis system contains: 40-70 g / L phenol, 50-90 g / L sodium pyruvate, 20-60 g / L ammonium chloride, 0.05-0.2 g / L pyridoxal phosphate, and 10-40 g / L fermentation cells. The temperature of the catalytic synthesis system is 30-45℃ and the pH is 7.0-10.

0.

6. The method as described in claim 5, characterized in that, In step S2, the fermentation cells of engineered tyrosine ammonia-lyase bacteria are used as catalysts. The concentration of L-tyrosine in the catalytic synthesis system is 40-100 g / L, and the concentration of fermentation cells is 10-40 g / L. The temperature of the catalytic synthesis system is 30-50℃, and the pH is 8.0-11.

0.

7. The method as described in claim 1, characterized in that, The tyrosine phenol lyase product and the tyrosine ammonia-lysin product were simultaneously added to the catalytic synthesis system; The tyrosine phenol lyase product is derived from the fermentation product of engineered tyrosine phenol lyase bacteria, and is the fermentation cell, fermentation broth, or tyrosine phenol lyase extracted from the fermentation cell or fermentation broth of engineered tyrosine phenol lyase bacteria; the tyrosine ammonia-lyase product is derived from the fermentation product of engineered tyrosine ammonia-lyase bacteria, and is the fermentation cell, fermentation broth, or tyrosine ammonia-lyase extracted from the fermentation cell or fermentation broth of engineered tyrosine ammonia-lyase bacteria. Alternatively, the tyrosine phenol lyase product and the tyrosine ammonia-lyase product are derived from the fermentation product of engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase, and are fermentation cells, fermentation broth, or tyrosine phenol lyase and tyrosine ammonia-lyase extracted from fermentation cells or fermentation broth of engineered bacteria that co-express tyrosine phenol lyase and tyrosine ammonia-lyase.

8. The method as described in claim 7, characterized in that, The fermentation method for the engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase is as follows: The seed culture of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase was inoculated into the fermentation medium. The fermentation system was controlled at a temperature of 32-42℃, a pH of 6.5-7.5, and dissolved oxygen above 30%. During fermentation, glycerol was continuously added at a rate of 100-150 mL / h to control the residual glycerol concentration in the fermentation system at 0.5-2 g / L and the cell growth rate at OD. 600 =3-4 / h; OD of bacterial cell concentration 600 When the temperature reaches 120°C or higher, the fermentation broth temperature is lowered and the cells are collected.

9. The method as described in claim 7, characterized in that, include: Using phenol, pyruvate, and ammonium chloride as substrates, and fermentation cells of engineered bacteria co-expressing tyrosine phenol lyase and tyrosine ammonia-lyase as catalysts, p-coumaric acid was synthesized.

10. The method as described in claim 9, characterized in that, The catalytic synthesis system contains fermentation cells of engineered bacteria co-expressing phenol, sodium pyruvate, ammonium chloride, pyridoxal phosphate, tyrosine phenol lyase, and tyrosine ammonia-lyase at a concentration of 40-70 g / L; The temperature of the catalytic synthesis system is 30-45℃ and the pH is 7.0-10.0.

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