Method for catalytically synthesizing tyramine by using tyrosine decarboxylase

Abstract

The invention discloses a method for preparing tyramine, which comprises the following steps that tyrosine is used as a substrate, in the presence of pyridoxal phosphate, tyrosine decarboxylase SEQ ID NO: 1 derived from streptococcus thermophilus or mutants SEQ ID NO: 3-5 of tyrosine decarboxylase SEQ ID NO: 1 are used for catalyzing decarboxylation reaction, tyramine is obtained, and the method has industrial development value.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme catalysis, and specifically relates to a tyrosine decarboxylase and its application in tyramine synthesis. Background Art

[0002] Tyramine (TA), also known as 4-hydroxyphenethylamine, p-hydroxyphenethylamine, p-phenol, caseamine, caseatoxin, and tyramine, is a compound synthesized in the normal metabolism of plants, insects, microorganisms, and animals. Its structural formula is shown below.

[0003]

[0004] Tyramine has a variety of physiological functions, including promoting metabolism, regulating blood sugar, blood pressure, and blood lipids, and enhancing immune capacity. It is currently widely used in pharmaceuticals and health products. At the same time, tyramine and its derivatives can also effectively reduce the production of melanin, so they are also used in beauty products.

[0005] The reported methods for preparing tyramine include chemical synthesis, biological fermentation, and biocatalysis. The chemical synthesis method has cheap raw materials and a relatively high recovery rate, which is suitable for industrial production and is currently the main method for tyramine synthesis. However, it generally has the following deficiencies: the synthesis route is complex, there are many reaction steps, the product purification is difficult, resulting in a low total yield, it requires corrosive raw materials such as strong acids and strong bases, which is harmful to the environment, and some require expensive metal catalysts and high-temperature and high-pressure equipment, increasing the production cost and operation risk. Studies have found that tyramine can be produced by the tyrosine metabolic pathway in vivo. The tyramine content in plant and insect cells is relatively low, and it is difficult to obtain high-purity tyramine by separation and extraction. However, the tyramine content and tyrosine decarboxylase activity in microbial cells are relatively high. Therefore, microorganisms can be selected for tyramine fermentation. Garai et al. studied Lactobacillus brevis CECT 216 isolated from cider, which could use a decarboxylase-specific medium. Under the conditions of 5% CO2 (v / v) and 28 °C, the tyramine yield was 854 mg / L after 7 days of fermentation (Garai G, Duenas MT, et al., 2007, 45(5): 473-478). Thakur et al. obtained 11.8 mg / L of tyramine after 16 h of shaking flask fermentation of Lactococcus lactis in a medium containing 0.2% tyrosine at 30 °C (Thakur M, Azmi W. 2009, 56(1): 101-114). Compared with the chemical synthesis method, the biological fermentation method has mild conditions, simple operation, and few by-products, but has the disadvantages of a long fermentation period, high medium cost, low yield, and difficulty in separating and extracting the product tyramine.

[0006] Tyrosine decarboxylase (TDC or TyDC) can catalyze the decarboxylation of tyrosine to produce tyramine and CO2. The research group of Ni Ye at Jiangnan University analyzed the protein structure of TDC from Lactobacillus brevis. Through the optimization of reaction conditions and molecular modification, when the tyrosine concentration was 400 mmol·L -1 , the conversion rate exceeded 99.99% after 24 h of reaction. Enzymatic catalysis has the characteristics of relatively specific products and can achieve one-step biotransformation synthesis. Therefore, there are certain advantages in preparing tyramine by the biocatalytic method. CN105969755A reported that the tyrosine decarboxylase and its S586A mutant from Lactobacillus brevis CGMCC1.2028 catalyzed the decarboxylation of tyrosine to obtain tyramine; CN115678877A reported that the tyrosine decarboxylase CtTyDC from Cistanche tubulosa catalyzed the decarboxylation of tyrosine to obtain tyramine, and the reaction occurred without relying on cofactors (pyridoxal phosphate, PLP). However, the disadvantage of these tyrosine decarboxylases is that the applicable substrate concentration is low, and there may be substrate / product inhibition, resulting in difficulty in industrial application, and the economic cost of tyramine production is difficult to compete with chemical synthesis methods. Summary of the Invention

[0007] In order to overcome the above defects of the existing enzymatic method for producing tyramine, the inventors are committed to mutating the reported tyrosine decarboxylases that can catalyze the decarboxylation of tyrosine to synthesize tyramine and screening for new varieties of tyrosine decarboxylases. Fortunately, a tyrosine decarboxylase that can tolerate high substrate concentrations was screened. It is derived from Streptococcus thermophilus. Through mutation technology and high-throughput screening, the enzyme activity was further improved, and it has the potential to be applied to the industrial production of tyramine. Specifically, the present invention includes the following technical solutions.

[0008] A method for preparing tyramine, characterized by comprising the following steps: using tyrosine as a substrate, in the presence of pyridoxal phosphate (PLP) as a coenzyme, using the tyrosine decarboxylase (Uniport ID accession number: E7AXL4; NCBI accession number: FR682467.1, named TDC3 herein) derived from Streptococcus thermophilus with the amino acid sequence shown in SEQ ID NO: 1 or its mutant to catalyze the decarboxylation reaction to obtain tyramine, wherein the mutant is a polypeptide with more than 90%, more than 92%, more than 95%, preferably more than 96%, preferably more than 97%, preferably more than 98%, more preferably more than 99% homology with the amino acid sequence of SEQ ID NO: 1 and with improved enzyme activity compared to SEQ ID NO: 1,

[0009]

[0010] In some embodiments, the terms "increased" or "enhanced" when referring to enzyme activity may mean an increase of about 50%, or at least about 80%, or at least about 90%, at least about 1-fold, or at least about 2-fold, or at least about 3-fold compared to a reference level such as the wild enzyme level.

[0011] Preferably, the above mutants are selected from the group consisting of: the N162Q mutant with an amino acid sequence as shown in SEQ ID NO: 3, the S243G mutant with an amino acid sequence as shown in SEQ ID NO: 4, and the A330Y mutant with an amino acid sequence as shown in SEQ ID NO: 5.

[0012] In the above reaction system, the concentration of the substrate tyrosine can be 50 - 100 g / L.

[0013] Among them, the addition amount of pyridoxal phosphate in the reaction system is 0.05 - 0.5 g / L, for example, about 0.1 g / L.

[0014] The reaction temperature of the above decarboxylation reaction is 20 - 55 °C, for example, 22 - 45 °C, preferably 25 - 35 °C, and preferably about 30 °C.

[0015] The above reaction can be carried out in a buffer solution such as phosphate buffer or acetate buffer, and the pH value of the reaction system is pH 4 - 7, preferably pH 4.5 - 6.5, and more preferably about pH 5.

[0016] It should be understood that when expressing numerical characteristics herein, the term "about" or "around" means that the indicated value can have an error range or floating range of ±10%, ±9%, ±8%, ±6% or ±5%.

[0017] Another aspect of the present invention provides a tyrosine decarboxylase mutant, which is a polypeptide selected from the following: the polypeptide with an amino acid sequence as shown in SEQ ID NO: 3, namely the N162Q mutant, the polypeptide with an amino acid sequence as shown in SEQ ID NO: 4, namely the S243G mutant, and the polypeptide with an amino acid sequence as shown in SEQ ID NO: 5, namely the A330Y mutant.

[0018] The present invention also provides a gene encoding the above tyrosine decarboxylase.

[0019] Preferably, the gene encoding the tyrosine decarboxylase mutant SEQ ID NO: 3 can be a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 6, or a polynucleotide having more than 90%, preferably more than 92%, preferably more than 95%, preferably more than 97%, preferably more than 98%, and more preferably more than 99% identity with SEQ ID NO: 6;

[0020] The gene encoding the tyrosine decarboxylase mutant SEQ ID NO:4 can be the polynucleotide shown in nucleotide sequence SEQ ID NO:7, or a polynucleotide having a sequence identity of more than 90%, preferably more than 92%, preferably more than 95%, preferably more than 97%, preferably more than 98%, more preferably more than 99% with SEQ ID NO:7;

[0021] The gene encoding the tyrosine decarboxylase mutant SEQ ID NO:5 can be the polynucleotide shown in nucleotide sequence SEQ ID NO:8, or a polynucleotide having a sequence identity of more than 90%, preferably more than 92%, preferably more than 95%, preferably more than 97%, preferably more than 98%, more preferably more than 99% with SEQ ID NO:8.

[0022] Another aspect of the present invention provides a DNA molecule comprising the above mutant-encoding gene.

[0023] Another aspect of the present invention provides a plasmid comprising the above DNA molecule. For example, the above plasmid can be a pET vector such as pET22b, pET24a, pET28a, or other commonly used vectors such as pSH plasmid.

[0024] Another aspect of the present invention provides a microorganism for expressing one of the above tyrosine decarboxylase mutants SEQ ID NOs:3-5, which integrates one of the above encoding genes SEQ ID NOs:6-8 in the host genome, or is a microorganism transformed with the above plasmid.

[0025] The transformation of the above plasmid can be carried out by conventional chemical transformation methods or electroporation methods into competent cells. The above gene editing techniques are selected from the following groups: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0026] Preferably, the above microorganism is a microorganism with a fast growth rate and suitable for expressing foreign recombinant proteins, such as selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida magnoliae, Pichia pastoris, Saccharomyces cerevisiae. The preferred microorganism is Escherichia coli, and more preferably Escherichia coli BL21(DE3).

[0027] Another aspect of the present invention provides the use of the above tyrosine decarboxylase mutant or the above microorganism in the production of tyramine.

[0028] Obviously, the above-mentioned tyrosine decarboxylase or the above-mentioned microorganism can be used as a biocatalyst for the enzymatic production of tyramine.

[0029] The tyrosine decarboxylase and its mutants derived from Streptococcus thermophiles discovered in the present invention have high tolerance to substrate concentration and can tolerate relatively high reaction temperatures. They can efficiently catalyze the production of tyramine from tyrosine and can completely convert 100 g / L of tyrosine into tyramine within 40 h. Description of the Drawings

[0030] Figure 1 It is a map of plasmid pET28a-TDC3 for expressing wild-type tyrosine decarboxylase TDC3 (SEQ ID NO:1).

[0031] Figure 2 It is an HPLC chromatogram of the strain BL21(DE3) / pET28a-TDC3 expressing wild-type tyrosine decarboxylase (SEQ ID NO:1) catalyzing the synthesis of tyramine from tyrosine. Detailed Description of the Invention

[0032] The bottleneck in the industrial application of enzymatic production of fine chemicals often lies in the substrate / product inhibition of enzymes. Although some technical means such as chemical modification of enzyme proteins and immobilization technology can overcome this defect to a certain extent, it will increase the cost of the catalyst and has no economic advantage. Using enzymes or mutants with high substrate / product tolerance can fundamentally solve this technical problem.

[0033] Therefore, based on biological big data, we have studied and screened tyrosine decarboxylases from numerous microbial sources, including Microbulbifer thermotolerans (named TDC1 in this article), Methanosarcina thermop (named TDC2 in this article), Streptococcus thermophiles (named TDC3 in this article), Clostridium thermobutyricum (named TDC4 in this article), Aspergillus thermomutatus (named TDC5 in this article), Aneurinibacillus thermoaerophilus (named TDC6 in this article), Enterococcus faecalis (named TDC7 in this article), Psilocybe cubensis (named TDC8 in this article), etc. Through experiments, it was found that the tyrosine decarboxylase from Streptococcus thermophiles (Uniport ID accession number: E7AXL4; NCBI accession number: FR682467.1, named TDC3 in this article) had relatively high enzyme activity and was tolerant to a relatively high substrate tyrosine concentration. The free wild enzyme could catalyze the reaction of tyrosine at a concentration of 100 g / L.

[0034] Modifying the original wild enzyme sequence through mutation technology is the main means to further improve enzyme activity and even enhance other properties such as environmental (temperature, pH) tolerance. We also mutated the wild-type tyrosine decarboxylase SEQ ID NO:1 and screened mutants SEQ ID NOs: 3 - 5 with significantly improved enzyme activity.

[0035] In this article, the terms "wild (type) enzyme", "original (type) enzyme", and "starting enzyme" have the same meaning, all referring to the tyrosine decarboxylase from Streptococcus thermophiles with the amino acid sequence of SEQ ID NO:1. Sometimes, for the sake of convenience in expression, in this article, the wild enzyme and its mutants such as SEQ ID NOs: 3 - 5, etc. can be collectively referred to as "tyrosine decarboxylase".

[0036] In this article, the above-mentioned term "(enzyme activity) improvement" or "increase" means an increase of at least 100% compared to the reference level, for example, an increase of at least about 1-fold, at least about 2-fold, or at least about 3-fold, or at least about 5-fold, or at least about 10-fold compared to the reference level.

[0037] The "mutation" described above includes, but is not limited to, substitution, deletion, insertion, and chemical modification of amino acid residues, preferably positive mutations, i.e., mutations that increase enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of non-conservative and conservative substitutions. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and thus generally includes substituting an amino acid in a polypeptide with an amino acid in the same or a similar amino acid-defined category. However, as used herein, if a conservative mutation can alternatively be an aliphatic-to-aliphatic, non-polar-to-non-polar, polar-to-polar, acidic-to-acidic, basic-to-basic, aromatic-to-aromatic, or restricted residue-to-restricted residue substitution, then a conservative mutation does not include hydrophilic-to-hydrophilic, hydrophobic-to-hydrophobic, hydroxyl-containing-to-hydroxyl-containing, or small residue-to-small residue substitutions. As is well known in the art, common cases of conservative substitutions include: the mutual substitution between aromatic amino acids F, W, and Y; the mutual substitution between hydrophobic amino acids L, I, and V; the mutual substitution between polar amino acids Q and N; the mutual substitution between basic amino acids K, R, and H; the mutual substitution between acidic amino acids D and E; the mutual substitution between amino acids with hydroxyl groups S and T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions are, for example:

[0038]

[0039] A "non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain characteristics. Non-conservative substitutions can use amino acids between rather than within the defined groups listed above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substitution region (e.g., proline substituting for glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0040] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while retaining enzyme activity and / or retaining the improved characteristics of the engineered aldolase. Deletions can be targeted to the interior and / or ends of the polypeptide. In multiple embodiments, deletions can include contiguous segments or can be discontinuous.

[0041] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the modified engineered aldolase includes inserting one or more amino acids into a naturally occurring aldolase and inserting one or more amino acids into other modified aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl or amino terminus. As used herein, insertions include fusion proteins as are known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0042] The amino acid numbers of the tyrosine decarboxylase mutants SEQ ID NOs: 3-5 of the present invention are 621 and the structures are clear. Therefore, those skilled in the art can easily obtain their coding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmid. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well-known to those skilled in the art.

[0043] In order to optimally express tyrosine decarboxylase in Escherichia coli, which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon-optimized. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms typically show a particular preference for one of several codons encoding the same amino acid due to mutation bias and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimized codons reflect the composition of their respective genomic tRNA pools. Thus, in fast-growing microorganisms, low-frequency codons for an amino acid can be replaced with high-frequency codons for the same amino acid. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0044] For example, in order to express tyrosine decarboxylase in Escherichia coli, the coding gene of the codon-optimized tyrosine decarboxylase mutant SEQ ID NO: 3 can be SEQ ID NO: 6; the coding gene of the tyrosine decarboxylase mutant SEQ ID NO: 4 can be SEQ ID NO: 7; the coding gene of the tyrosine decarboxylase mutant SEQ ID NO: 5 can be SEQ ID NO: 8.

[0045] The above-mentioned transformant hosts can be any microorganism suitable for expressing tyrosine decarboxylase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21(DE3).

[0046] When used as a biocatalyst for the production of tyramine, the tyrosine decarboxylase of the present invention can be in the form of an enzyme or a bacterial cell. The form of the enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, enzyme immobilized on a carrier, etc.; the form of the bacterial cell includes viable bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0047] When microorganisms such as Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli are no longer used for fermentation and proliferation but for enzymatic catalysis reactions, they are a natural immobilized enzyme in themselves, and without the need for cell disruption treatment, or even extraction and purification treatment, they can be used as an enzyme preparation for catalytic reactions. Since both the reaction substrate and the reaction product are small molecule compounds, they can easily pass through the biological barrier of the bacterial cell - the cell membrane. Therefore, there is no need to perform cell disruption treatment on the bacterial cells, which is economically beneficial.

[0048] Pyridoxal phosphate (PLP) can also be added as a coenzyme to the reaction system of the present invention. Pyridoxal phosphate acts as a coenzyme in many enzymatic processes, and its functions include decarboxylation, deamination, transamination, racemization, etc. In the present invention, it mainly plays a role in decarboxylation, which helps to promote the conversion of tyrosine to tyramine.

[0049] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0050] Embodiment

[0051] In this article, the addition amounts, contents and concentrations of various substances are involved. Among them, the percentage content, unless otherwise specified, all refer to the mass percentage content.

[0052] Materials and Methods

[0053] The total gene synthesis, primer synthesis and sequencing in the examples were all completed by Genewiz (Suzhou) Inc.

[0054] The molecular biology experiments in the examples, including plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, culture medium preparation, etc., mainly referred to "Molecular Cloning: A Laboratory Manual" (Third Edition), edited by J. Sambrook, D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0055] The PCR amplification experiments were carried out according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments when necessary.

[0056] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (For LB solid medium, add 20 g / L agar powder additionally.)

[0057] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0 - 7.5. (For TB solid medium, add 20 g / L agar powder additionally.)

[0058] 20X electrotransformation mother liquor: 80 g / L glycine, 2% Tween 80

[0059] Max DNA Polymerase: Takara Biotechnology Co., Ltd.

[0060] FastDigest DpnI: Thermo Scientific Biotechnology Co., Ltd.

[0061] Tyrosine: Shanghai Macklin Biochemical Co., Ltd.

[0062] Pyridoxal phosphate: Shanghai Macklin Biochemical Co., Ltd.

[0063] Tyramine standard: Shanghai Macklin Biochemical Co., Ltd.

[0064] It should be noted that for the convenience of description, in the examples, the strain number, plasmid number, enzyme number, and enzyme - encoding gene number can share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0065] Example 1: Construction of recombinant Escherichia coli engineering bacteria expressing tyrosine decarboxylase

[0066] 1. Construction of engineering bacteria expressing tyrosine decarboxylase TDC3 derived from Streptococcus thermophiles

[0067] 1.1 According to the amino acid sequence of tyrosine decarboxylase from Streptococcus thermophiles (Uniport ID accession number E7AXL4, named TDC3 in this article), codon optimization was carried out according to the codon preference of Escherichia coli to obtain its coding gene SEQ ID NO:2. It was entrusted to Suzhou GeneScript Biotech Co., Ltd. for total gene synthesis and cloned into plasmid pET28a. It was inserted into the XhoI site at the 3' end and seamlessly cloned at the 5' end to obtain plasmid pET28a - TDC3. See Figure 1 。

[0068] 1.2 The recombinant plasmid pET28a-TDC3 was transformed into the competent Escherichia coli BL21(DE3) by electroporation to obtain the engineered E. coli strain BL21(DE3) / pET28a-TDC3.

[0069] 2. According to the same method as in step 1 above, the engineered E. coli strains BL21(DE3) / pET28a-TDC1, BL21(DE3) / pET28a-TDC2, BL21(DE3) / pET28a-TDC4, BL21(DE3) / pET28a-TDC5, BL21(DE3) / pET28a-TDC6, BL21(DE3) / pET28a-TDC7, and BL21(DE3) / pET28a-TDC8 expressing tyrosine decarboxylase from Microbulbifer thermotolerans, Methanosarcina thermop, Clostridium thermobutyricum, Aspergillus thermomutatus, Aneurinibacillus thermoaerophilus, Enterococcus faecalis, and Psilocybe cubensis were constructed respectively. The information of each enzyme and strain number is listed in Table 1.

[0070] Table 1. Sources of tyrosine decarboxylase and information of engineered strains

[0071]

[0072] Example 2: Expression of engineered strains of recombinant tyrosine decarboxylase and preparation of pure enzyme

[0073] The recombinant strains obtained in Example 1 were induced for expression to obtain pure enzymes of each strain. The operating steps are as follows:

[0074] 2.1 Each engineered strain was picked from the LB plate into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight for 12 h in a shaker at 37°C and 220 rpm. Then, it was inoculated into the LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1% v / v and cultured at 37°C until the OD 600 reached 0.6 - 0.8. IPTG with a final concentration of 0.5 mmol / L was added, and after inducing expression for 12 h in a shaker at 18°C and 220 rpm, the cells were centrifuged at 4°C and 4000 rpm for 10 min to collect the cells (i.e., wet cells).

[0075] 2.2 Resuspend the collected bacterial cells with 20 mL of equilibration buffer (50 mM HEPES, 50 mM NaCl, 10 mM imidazole, pH 7.5) to obtain a cell suspension. Perform ultrasonic disruption on the cell suspension under ice bath conditions to obtain a crude enzyme solution. Centrifuge the crude enzyme solution at 12,000 rpm for 30 min to collect the supernatant, and use a nickel column to extract the pure enzyme. First, rinse the nickel column with water, then rinse and equilibrate the nickel column with 10 column volumes of equilibration buffer. Load the supernatant and allow it to bind to the packing material for a period of time. Elute the impurity proteins with 20 mL of washing solution (50 mM HEPES, 50 mM NaCl, 25 mM imidazole, pH 7.5), and add 4 mL of elution solution (50 mM HEPES, 50 mM NaCl, 250 mM imidazole, pH 7.5) to elute the target protein. Aliquot 1 mL into 1.5 mL EP tubes, which is the pure enzyme solution. After the elution is completed, first rinse the nickel column with 10 column volumes of 2 M imidazole, then rinse with 10 column volumes of water, and finally add 20% ethanol solution and store it in a refrigerator at 4°C. Use Bradford method to measure the protein concentration: Add 1 mL of Bradford to 30 μL of protein (determine the dilution factor according to the blue color intensity, dilute with PBS), use the addition of PBS as the control group, take out 150 μL and transfer it to an enzyme-linked immunosorbent assay (ELISA) plate, and detect it at a wavelength of 595 nm. The expression levels of each recombinant enzyme in the eluate are listed in Table 2. Freeze-dry the pure enzyme solution of each enzyme under vacuum to obtain the enzyme protein, and store it in a refrigerator at 4°C for standby.

[0076] Table 2. TDC protein concentrations in the column eluates of each strain

[0077]

[0078] As can be seen from Table 2, there are significant differences in the amounts of enzyme proteins expressed by different engineered bacteria. Among them, the expression levels of TCD2, TCD3, and TCD5 are relatively high; the expression levels of TDC1, TDC4, and TDC8 proteins are relatively low.

[0079] Example 3: Examine the enzyme activity of tyrosine decarboxylase from different sources in catalyzing tyrosine

[0080] Using the pure enzyme extracted in Example 2 as the reaction catalyst, the reaction volume is 1 mL, the enzyme protein concentration is 1 mg / mL, the tyrosine concentration is 30 g / L, and the addition amount of pyridoxal phosphate aqueous solution is 0.1 g / L. After reacting at 25°C, 300 rpm, and pH 5.0 for 20 h, perform high-performance liquid chromatography (HPLC) detection.

[0081] HPLC detection conditions: Agilent 1260 high-performance liquid chromatograph, chromatographic column: Agilent Eclipse SB-Aq column; column temperature: 25°C; mobile phase: 0.75% sodium dihydrogen phosphate (pH = 3.7): acetonitrile = 90:10 (volume ratio); flow rate: 1 mL·min -1; Detector: UV detector; Detection wavelength: 210 nm; Injection volume: 1 μL.

[0082] The relative enzyme activity of tyrosine decarboxylase was determined by comparing the concentration of the product tyramine, i.e., the yield, or the conversion rate of the raw material tyrosine.

[0083] Comparative experiments found that under the condition of pH 5.0, the enzyme activities of the two tyrosine decarboxylases TDC3 and TDC7 were relatively high, and their catalytic abilities were generally equivalent, both being at least 80% higher than those of the other six tyrosine decarboxylases TDC1, TDC2, TDC4, TDC5, TDC6, and TDC8.

[0084] Based on the comprehensive consideration of enzyme activity and the expression level of the engineered bacteria, TDC3 was mainly studied in detail.

[0085] Example 4: Enzyme activity of TDC3 catalyzing tyrosine at different pH values

[0086] The pH adaptability of TDC3 was investigated. Using the pure enzyme TDC3 prepared by extraction in Example 2 as the reaction catalyst, the reaction volume was 1 mL, the enzyme protein concentration was 1 mg / mL, the tyrosine concentration was 30 g / L, and the addition amount of pyridoxal phosphate aqueous solution was 0.1 g / L. After reacting at 25 °C and 300 rpm for 20 h under the conditions of pH 4.5 and pH 6.5 respectively, HPLC detection was carried out.

[0087] The catalytic activities, i.e., the enzyme activities, of TDC3 catalyzing tyrosine to produce tyramine under the two pH values are listed in Table 2.

[0088] Table 2. Detection results of the enzyme activity of TDC3 catalyzing tyrosine to produce tyramine under two pH values

[0089]

[0090] It can be seen from Table 2 that under the conditions of pH 4.5 - 6.5, the catalytic abilities of the tyrosine decarboxylase TDC3 are generally equivalent.

[0091] Example 5: Catalytic activity of TDC3 catalyzing tyrosine at different temperatures

[0092] The environmental temperature adaptability of TDC3 was further investigated. The reaction volume was 1 mL, the TDC3 protein concentration was 1 mg / mL, the tyrosine concentration was 30 g / L, and the addition amount of pyridoxal phosphate aqueous solution was 0.1 g / L. Under the condition of pH 4.5, the reactions were carried out at 35 °C, 45 °C, and 55 °C for 20 h respectively, and the samples were analyzed by HPLC. The catalytic activities of tyrosine decarboxylase TDC3 at the three temperatures are listed in Table 3.

[0093] Table 3. Detection results of TDC3 catalyzing tyrosine to produce tyramine at different temperatures

[0094]

[0095] As can be seen from Table 3, the enzymatic activities of TDC3 at the three temperatures are not very different. The reaction rate is the fastest at 45 °C, and it can still catalyze the reaction without inactivation at the relatively high temperature of 55 °C, indicating that TDC3 has high thermal stability.

[0096] Example 6: Preparation of tyramine by catalyzing tyrosine with TDC3 cells

[0097] In this example, the fermented cells were used as biocatalysts to investigate the specific situation of directly catalyzing the reaction of the substrate tyrosine.

[0098] 6.1 Preparation of wet recombinant Escherichia coli cells containing TDC3

[0099] Considering that the internal environment of the cells is conducive to maintaining the enzyme activity and the adaptability of the enzyme to the environment (pH, temperature), the wet cells containing TDC3 were obtained according to the steps in Example 2, and stored at -20 °C overnight for later use.

[0100] 6.2 Preparation of 20 mM acetate buffer as the reaction solution

[0101] The decarboxylation reaction of tyrosine will produce CO2, which will increase the pH of the reaction solution and is not conducive to the enzymatic reaction process. Using a buffer solution system is beneficial to controlling the stability of the reaction system pH and is conducive to the enzyme or wet cells to catalyze the synthesis of tyramine.

[0102] The reaction buffer can be a phosphate buffer solution or an acetate buffer solution. For example, it is 20 mM acetate buffer (pH = 5.0). In a 10 mL reaction system, 100 g / L tyrosine, 10 g / L cells, and 0.34 g / L PLP are added. During the process, acetic acid is used to control the pH during the reaction not to exceed 5.0. The reaction is carried out at 40 °C and 300 rpm for 2 h, and samples are taken for liquid phase analysis. After 2 h of reaction, the tyramine yield reaches 97%.

[0103] 6.3 Investigation of the catalytic synthesis of tyramine when the reaction solution is water

[0104] When water is used as the reaction solution, the reaction pH can be regulated with dilute hydrochloric acid / sodium hydroxide solution. When the cell addition amount is 30 g / L, the 100 g / L tyrosine can be completely converted after reacting for 40 h.

[0105] Example 7: Attempts at enzyme mutation

[0106] In order to improve the enzyme activity of TDC3, we also tried to modify TDC3 by mutation and high-throughput screening methods. The construction of the single-site saturation mutation library of tyrosine decarboxylase includes the following steps.

[0107] Saturated mutagenesis libraries at different sites of tyrosine decarboxylase TDC3 were constructed separately. The mutagenesis libraries were obtained by screening the constructed mutant libraries using degenerate primers. Using the plasmid pET28a-TDC3 expressing TDC3 in Example 1 as a template, and applying the primers designed as shown in Table 4, Prime Max DNA Polymerase from Takara Biotechnology Co., Ltd. was used for PCR amplification to obtain the corresponding linearly mutated plasmid vectors.

[0108] The PCR system was as follows: 1 μL of template, 2 μL each of the upstream and downstream primers, 25 μL of Max DNA Polymerase, and 20 μL of double-distilled water.

[0109] The amplification program was: 1 cycle: 98 °C for 30 s; 30 cycles: 98 °C for 10 s; 55 °C for 10 s; 72 °C for 3 min; 1 cycle: 72 °C for 5 min.

[0110] After the PCR reaction was completed, 25 μL of the PCR amplification product was taken, 2 μL of DpnI was added, 3 μL of 10×FastDigest buffer was added, and it was incubated at 37 °C for 2 h. Subsequently, it was transformed into competent Escherichia coli BL21(DE3) cells. 10 μL of the digested product was added to 100 μL of E. coli BL21(DE3) competent cells, and the tube wall was flicked several times to mix evenly, and it was placed in an ice-water bath for 30 min. Heat shock at 42 °C for 45 s and incubate on ice for 3 min. 900 μL of LB medium without antibiotics was added, and it was incubated at 37 °C for 60 min. 200 μL of the bacterial solution was taken and evenly spread on an LB plate containing 50 μg / mL kanamycin. The plate was inverted and cultured overnight at 37 °C. 10 strains of bacteria were randomly selected from each plate, and Suzhou Genewiz Biotechnology Co., Ltd. was entrusted to perform genomic sequencing and alignment on the strains to determine the changes in their amino acid sequences and check whether the mutation rate reached the requirements, so as to facilitate the selection of strains with significantly improved enzyme activity.

[0111] Table 4. Primer sequences used for constructing the saturated library

[0112]

[0113]

[0114] F: Forward primer; R: Reverse primer.

[0115] Example 8: Expression and screening of the saturated mutagenesis library

[0116] Inoculate the wild-type enzyme-expressing strain and mutant strain into a 96-well plate containing 700 μL of auto-induction medium per well, and culture overnight at 30 °C. One 96-well plate is inoculated with the library at each site to obtain the fermentation broth. Centrifuge at 3600 rpm for 10 min, remove the supernatant, and store the cells at -20 °C overnight for high-throughput screening.

[0117] The above auto-induction medium contains 935 mL of ZY basal medium (10 g peptone, 5 g yeast extract, made up to 935 mL), 40 mL of 25×M (1.25 M Na2HPO4·12H2O, 1.25 M KH2PO4, 1.25 M NH4Cl, 1.25 M Na2SO4), 20 mL of 50×5052 (125 g glycerol, 12.5 g glucose, 50 g α-lactose), 2 mL of 1 M MgSO4, 2 mL of 1000× trace elements (FeCl3·6H2O, CaCl2·2H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O, CuCl2·2H2O, NiCl2·6H2O, Na2MoO4·2H2O, Na2SeO3, H3BO3), and kanamycin at a final concentration of 50 μg / mL. The culture conditions are 30 °C and 250 rpm.

[0118] The above high-throughput screening method is as follows: Prepare 20 mM acetate buffer (pH = 5.0). Resuspend the above frozen wet cell mass with 600 μL of water. The 1 mL reaction system includes 975 μL of 20 mM acetate buffer, 100 mM tyrosine, 0.1 g / L PLP, and 20 μL of wet cells. After mixing evenly, take out 500 μL of the reaction solution and add it to a new 96-well plate. Add 5 μL of 2.5 mM phenol red and react at 40 °C and 250 rpm for 2 h. Centrifuge the reaction solution, take 150 μL of the supernatant and transfer it to an enzyme-linked immunosorbent assay (ELISA) plate for scanning at OD573 nm. In acetate buffer (pH = 5), phenol red is yellow. When tyramine exists in the system, it indicates that the pH increases and the color changes from yellow to orange. The more obvious the color change within the same time, the higher the enzyme activity.

[0119] Through the screening of 15 saturated mutation libraries, based on the color development results and HPLC data, initially select 5 strains, namely 98-H4, 162-D12, 243-B1, 243-D2, and 330-D7, as potential positive mutant strains. Entrust Genewiz (Suzhou) Inc. to perform genomic sequencing and alignment of the strains to determine the amino acid sequence changes. After sequencing verification, 98-H4 and 243-D2 are wild-type and will not be subjected to re-screening.

[0120] Table 6. Sequencing results of some positive mutant strains

[0121]

[0122]

[0123] According to the method of Example 2, the wild-type enzyme-expressing strain BL21(DE3) / pET28a-TDC3 and the mutant strains 162-D12, 243-B1, and 330-D7 were cultured and induced for expression to obtain the pure enzymes of each strain.

[0124] According to the same method as in Example 4, the enzyme activities of TDC3, the N162Q mutant, the S243G mutant, and the A330Y mutant were compared.

[0125] The reaction volume was 1 mL, the enzyme protein concentration was 1 mg / mL, the tyrosine concentration was 60 g / L, the addition amount of pyridoxal phosphate aqueous solution was 0.1 g / L. Under the condition of pH 4.5, the reaction was carried out at 30 °C for 20 h, and the samples were analyzed by HPLC. The catalytic activities of the enzymes were compared by the concentration of the product tyramine. The relative enzyme activity of TDC3 was set as 100%, and the comparison results of the activities of the four enzymes are listed in Table 7.

[0126] Table 7. Comparison of enzyme activities between wild-type and mutants

[0127]

[0128] The above results show that the enzyme activities of the three mutants N162Q mutant (SEQ ID NO: 3), S243G mutant (SEQ ID NO: 4), and A330Y mutant (SEQ ID NO: 5) obtained by the mutation technique have been improved to varying degrees compared with the wild-type enzyme TDC3 (SEQ ID NO: 1). Among them, the enzyme activity of A330Y is the highest, and they can all tolerate a higher substrate concentration and have the potential to be used for catalyzing the production of tyramine from tyrosine.

Claims

1. A method for preparing tyramine, characterized in that, It includes the following steps: Using tyrosine as a substrate, in the presence of pyridoxal phosphate as a coenzyme, using the tyrosine decarboxylase derived from Streptococcus thermophiles (Uniport ID accession number: E7AXL4; NCBI accession number: FR682467.1) with the amino acid sequence shown in SEQ ID NO: 1 or its mutant to catalyze the decarboxylation reaction to obtain tyramine, wherein the mutant is a polypeptide with more than 90% homology to the amino acid sequence of SEQ ID NO: 1 and with increased enzyme activity compared to SEQ ID NO:

1.

2. The method according to claim 1, wherein The mutants are selected from the following group: the N162Q mutant with the amino acid sequence shown in SEQ ID NO: 3, the S243G mutant with the amino acid sequence shown in SEQ ID NO: 4, and the A330Y mutant with the amino acid sequence shown in SEQ ID NO:

5.

3. The method according to claim 1, wherein The concentration of the substrate tyrosine in the reaction system is 50 - 100 g / L.

4. The method according to claim 1, wherein The addition amount of pyridoxal phosphate in the reaction system is 0.05 - 0.5 g / L, for example, about 0.1 g / L.

5. A tyrosine decarboxylase mutant, which is a polypeptide selected from the following: the polypeptide with the amino acid sequence shown in SEQ ID NO: 3, namely the N162Q mutant, the polypeptide with the amino acid sequence shown in SEQ ID NO: 4, namely the S243G mutant, and the polypeptide with the amino acid sequence shown in SEQ ID NO: 5, namely the A330Y mutant.

6. A gene encoding the tyrosine decarboxylase mutant as claimed in claim 5; preferably, the gene encoding the tyrosine decarboxylase mutant SEQ ID NO: 3 is the polynucleotide shown in the nucleotide sequence SEQ ID NO: 6; the gene encoding the tyrosine decarboxylase mutant SEQ ID NO: 4 is the polynucleotide shown in the nucleotide sequence SEQ ID NO: 7; the gene encoding the tyrosine decarboxylase mutant SEQ ID NO: 5 is the polynucleotide shown in the nucleotide sequence SEQ ID NO:

8.

7. A DNA molecule, characterized in that, It contains the gene as claimed in claim 6.

8. A plasmid containing the DNA molecule as claimed in claim 7.

9. A microorganism for expressing the tyrosine decarboxylase mutant as described in claim 5, characterized in that, A microorganism with the gene as claimed in claim 6 integrated into its genome or transformed with the plasmid as claimed in claim 8.

10. Use of the tyrosine decarboxylase mutant as claimed in claim 5 or the microorganism as claimed in claim 9 in the production of tyramine.

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