Phenol-resistant tyrosine phenol-lyase mutants and use thereof

Abstract

The present application is found by screening that the tyrosine phenol-lyase from Dysgonomonas massiliensis ​ The tyrosine phenol-lyase mutant can maintain high enzyme activity under the condition of high concentration of phenol, and can be used to catalyze the reaction of phenol, pyruvic acid and ammonium salt to produce tyrosine, so that the reaction concentration of the substrate can be increased, the reaction speed can be accelerated, and the production efficiency of tyrosine can be effectively improved.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, specifically to a phenol-resistant tyrosine phenol lyase mutant and its applications. Background Technology

[0002] Tyrosine, also known as 2-hydroxy-3-p-hydroxyphenylpropionic acid, is an aromatic polar α-amino acid containing a phenolic hydroxyl group. It has applications in various fields such as pharmaceuticals, health products, food additives, and cosmetics. In the pharmaceutical industry, it is mainly used to synthesize important precursors such as dopamine and thyroxine. In the food industry, it can be used as a nutritional fortifier in special diets. With the world gradually entering an aging society and driven by health-conscious consumption trends, the demand for nutritional and health-promoting products like tyrosine, which have clearly defined effects, is increasing daily, indicating promising market potential.

[0003] The main methods for producing tyrosine include direct extraction, chemical synthesis, and biological methods. Direct extraction involves acid hydrolysis or enzymatic hydrolysis of natural proteins rich in tyrosine. This method is simple and requires minimal equipment, but suffers from low extraction efficiency, poor product purity, and volatile raw material prices. Chemical synthesis primarily uses petroleum derivatives as raw materials, generating DL-tyrosine through chemical reactions (such as the Erlenmeyer-Plöchl synthesis method), which is then separated to obtain naturally occurring L-tyrosine. This method uses inexpensive raw materials and has mature processes, but it causes significant environmental pollution, and the products are often racemic mixtures (DL-tyrosine), making separation difficult. Biological methods include microbial fermentation and biocatalysis. Microbial fermentation utilizes genetically engineered bacteria to metabolize sugars and directly synthesize L-tyrosine within the cells. While eliminating the influence of optical isomers, the complex metabolic systems of the producing strains make the fermentation process difficult to control, resulting in low acid production rates. Biocatalysis offers advantages such as high reaction specificity, high product purity, mild reaction conditions, ease of purification, and environmental friendliness, making it an effective, green, and inexpensive method for producing L-tyrosine.

[0004] Currently, the biocatalytic method for tyrosine synthesis is mainly based on the reaction of tyrosine phenol-lyase (TPL) catalyzing the formation of L-tyrosine from the substrates pyruvate, phenol, and ammonium. Since the presence of phenolic hydroxyl groups in phenolic substances, such as phenol, not only irreversibly destroys the active site of the enzyme but also disrupts the three-dimensional structure of the protein, leading to denaturation, the tolerance of the catalytic enzyme to phenolic substances often becomes a crucial factor determining key indicators such as yield and production rate.

[0005] Kim DY et al. (Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase. J Microbiol Biotechnol (2007 Jan;17(1):116-122. PMID: 18051362.) A fed-batch reactor was used to synthesize tyrosine in vitro with tyrosine phenol lyase as a biocatalyst. The concentration of phenol needed to be strictly controlled below 75 mM by continuously supplying key substrates such as phenol to reduce the inhibitory effect of phenol on the reaction. However, this method had a low production rate of only 24.1 mM / h. Guo et al. (A bi-enzymatic cascade to yield pyruvate as co-substrate for L-tyrosine production.) Appl. Microbiol Biotechnol (2020. 104(23): p. 10005-10018) Using alanine as a substrate, it is first converted to pyruvate through a double enzyme cascade reaction of alanine racemic enzyme and D-amino acid oxidase, and then tyrosine is produced by catalysis of tyrosine phenol lyase. The tyrosine phenol lyase used in this method is also intolerant to high concentrations of phenol. In order for the reaction to proceed normally, the concentration of phenol during the reaction process needs to be strictly controlled not to exceed 60 mM. It can be seen that the current strategies to solve the problem of phenol intolerance of tyrosine phenol lyase are mostly focused on controlling the concentration of phenol, reducing the initial concentration of phenol and controlling it to always be in a low concentration range by adding phenol at a low rate during the process. However, this strategy requires high detection sensitivity and control accuracy. Once the local concentration of phenol accumulates and exceeds the enzyme's tolerance threshold, it will lead to the inactivation of tyrosine phenol lyase and the reaction will be difficult to continue. In large-scale production, the precise control of phenol concentration has extremely high requirements for equipment and has a low fault tolerance. In addition, controlling the substrate concentration also limits the increase of the reaction rate, which seriously affects the production efficiency. It is evident that the strategy of controlling phenol concentration can only address the symptoms and does not fundamentally solve the problem of phenol inhibiting enzyme activity.

[0006] In addition to producing tyrosine, tyrosine phenol-lyase also exhibits broad substrate diversity, recognizing a variety of phenolic derivatives as substrates. By using phenols with the same or different substituents at the ortho and / or para and / or meta positions as substrates, it catalyzes the synthesis of a series of rare amino acid intermediates used in the manufacture of antibiotics, antiviral drugs, or anticancer drugs. (Zheng, R.-C. et al., "Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA") Enzyme and Microbial Technology (2018. 112: pp. 88-93) Using catechol instead of phenol as a substrate, L-DOPA was directly synthesized by reacting it with pyruvate and ammonia. Similarly, to maintain enzyme activity, the concentration of the substrate catechol was controlled below 45 mM during the reaction. It is evident that regardless of the reaction method, the fundamental problem of inhibition of tyrosine phenol lyase activity by the phenolic hydroxyl group exists.

[0007] To fundamentally solve the problem of tyrosine phenol lyase's intolerance to phenolic substrates, finding or modifying tyrosine phenol lyase with high phenol tolerance has become an urgent need for large-scale production. Summary of the Invention

[0008] One of the objectives of this invention is to provide a tyrosine phenol lyase with high enzyme activity.

[0009] Another objective of this invention is to provide a tyrosine phenol lyase mutant that can tolerate high concentrations of phenolic compounds and its applications.

[0010] To achieve the first objective of this invention, a source derived from... Dysgonomonas massiliensis Tyrosine phenol lyase (DmTPL) has high catalytic activity.

[0011] To achieve another objective of the present invention, in a first aspect, the present invention provides a tyrosine phenol lyase mutant resistant to high concentrations of phenolic compounds, said mutant being derived from... Dysgonomonas massiliensis Based on the amino acid sequence of tyrosine phenol lyase (DmTPL), it is obtained by mutating sites selected from the following groups or combinations thereof:

[0012] 1) The 84th amino acid is mutated from C to K;

[0013] 2) The 21st amino acid is mutated from M to R;

[0014] 3) The second amino acid is mutated from N to K;

[0015] 4) The 45th amino acid is mutated from D to K;

[0016] 5) The 372nd amino acid is mutated from N to A;

[0017] 6) The 84th amino acid is mutated from C to K, and the 21st amino acid is mutated from M to R.

[0018] The source Dysgonomonas massiliensis The amino acid sequence of the tyrosine phenol lyase is shown in SEQ ID NO:1.

[0019] The phenolic compound may be phenol or a phenol derivative.

[0020] The phenol derivative is preferably a compound derived from phenol with the same or different substituents at the ortho and / or meta and / or para positions.

[0021] In a second aspect, the present invention provides a nucleic acid molecule encoding the said tyrosine phenol lyase mutant.

[0022] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0023] Fourthly, the present invention provides the application of the tyrosine phenol lyase mutant, the nucleic acid molecule, or the biological material.

[0024] The application includes a reaction catalyzed by tyrosine lyase.

[0025] The preferred applications are those in tyrosine production and L-DOPA production.

[0026] Fifthly, the present invention provides a method for synthesizing tyrosine, using pyruvate or its salt, phenol, or ammonium salt as a substrate, and contacting the substrate with the tyrosine phenol lyase mutant to enzymatically synthesize tyrosine.

[0027] In a sixth aspect, the present invention provides a method for synthesizing L-DOPA, using pyruvate or its salt, catechol, or ammonium salt as substrates, and contacting the substrates with the tyrosine phenol lyase mutant to enzymatically synthesize L-DOPA.

[0028] In one specific embodiment of the present invention, in order to obtain the tyrosine phenol lyase mutant in the enzyme reaction system, Escherichia coli is used as the host cell, the tyrosine phenol lyase mutant is recombinantly expressed in Escherichia coli, the recombinant Escherichia coli is cultured, and the recombinant cells are crushed under high pressure to obtain a reaction enzyme solution containing the tyrosine phenol lyase mutant.

[0029] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0030] This invention has identified highly active [products] from [sources]. Dysgonomonas massiliensis The tyrosine phenol lyase (DmTPL) was mutated to obtain mutants TPL-C84K, TPL-M21R, and TPL-C84K-M21R, which are tolerant to high concentrations of phenol. In particular, the mutant TPL-C84K-M21R showed almost no decrease in enzyme activity at a high phenol concentration of 100 mM, and maintained high enzyme activity even at ultra-high phenol concentrations of 150 mM. Using this mutant to catalyze the reaction of phenol, pyruvate, and ammonium salt to produce tyrosine can increase the substrate concentration, accelerate the reaction rate, and effectively improve the production efficiency of tyrosine. Attached Figure Description

[0031] Figure 1 Relative enzyme activities of tyrosine phenol lyases from different sources

[0032] Figure 2 The relative enzyme activity of a single mutant of tyrosine phenol lyase at different phenol concentrations

[0033] Figure 3 The relative enzyme activity of the tyrosine phenol lyase double mutant at different phenol concentrations

[0034] Figure 4 Results of catalytic activity assay of tyrosine phenol lyase double mutant at high phenol concentrations Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, all experimental conditions are conventional conditions well known to those skilled in the art.

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or as recommended by the manufacturer's instructions.

[0037] Example 1: Enzyme activity screening of tyrosine phenol lyase TPL from different sources

[0038] (1) Based on literature review, sequence alignment and other methods, the following four tyrosine phenol lyases from different sources were screened: Dysgonomonas massiliensis Tyrosine phenol lyase (DmTPL), derived from Tissierellia bacteriaTyrosine phenol lyase (TbTPL), derived from Erysipelotrichia bacterium Tyrosine phenol lyase (EbTPL), derived from Bacteroidetes bacteria The amino acid sequences of the tyrosine phenol lyase (BbTPL) are shown in SEQ ID NO: 1-4.

[0039] (2) The above four different sources of tyrosine lyase were tested by Qingke Biotechnology Co., Ltd. E.coli After codon optimization, gene synthesis was performed (the optimized codon sequences are shown in SEQ ID NO: 5-8), and the gene was integrated into the pBAD plasmid vector to obtain recombinant plasmids pBAD-DmTPL, pBAD-TbTPL, pBAD-EbTPL, and pBAD-BbTPL, respectively. These plasmids were then transformed into BW25113 to obtain recombinant strains BW25113-DmTPL, BW25113-TbTPL, BW25113-EbTPL, and BW25113-BbTPL.

[0040] (3) The recombinant strain constructed above was inoculated into LB medium supplemented with the corresponding resistance and cultured with shaking at 37°C. When the bacterial concentration reached OD 600 of 0.6-0.8, arabinose with a final concentration of 2 g / L was added for induction. After culturing at 30°C for 20 h, wet bacterial cells were collected by centrifugation.

[0041] (4) Wash the bacterial cells with sterile water and centrifuge at 4°C to remove the supernatant. Repeat twice.

[0042] (5) Add lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0) to fully suspend the bacterial cells, break the bacterial cells under low temperature and high pressure, centrifuge at 4 °C, and collect the supernatant to obtain crude enzyme solution.

[0043] (6) Take crude enzyme solution and determine the relative enzyme activity of tyrosine phenol lyase from different sources under 50 mM low concentration phenol conditions.

[0044] Composition of enzyme activity assay reaction solution: Tris-HCl 100 mM, sodium pyruvate 50 mM, ammonium chloride 1 M, phenol 50 mM, crude enzyme solution (2 g of crude enzyme solution obtained by crushing wet bacterial cells per liter of reaction solution).

[0045] Reaction conditions: 35℃, pH 8.5, reaction time 30 min. After the reaction, the amount of tyrosine produced was determined by HPLC, and the relative enzyme activity of each enzyme was calculated.

[0046] The results are as follows Figure 1As shown, with the highest enzyme activity of DmTPL as 100%, the enzyme activities of TbTPL, BbTPL, and EbTPL were 68.1%, 46.9%, and 29.3%, respectively. This result indicates that the enzyme activity derived from DmTPL is 100%. Dysgonomonas massiliensis The tyrosine phenol lyase DmTPL has the highest enzyme activity and is expected to achieve a breakthrough in industrial production. Therefore, DmTPL was selected as the basis for constructing a phenol-resistant tyrosine phenol lyase mutant.

[0047] Example 2: Construction of a single mutant of tyrosine phenol lyase TPL

[0048] (1) Using pBAD-DmTPL as a template, the DmTPL gene was mutated at the corresponding sites using the mutation primers in Table 1. The PCR amplification products were then used... Dpn After digestion with enzyme I for 2 hours, the cells were transformed into E. coli DH5α competent cells. Single clones were selected and sequenced to identify plasmids containing mutant genes: pBAD-TPL-C84K, pBAD-TPL-M21R, pBAD-TPL-N2K, pBAD-TPL-D45K, and pBAD-TPL-N372A.

[0049] (2) The plasmids containing the mutant gene were transformed into Escherichia coli BW25113 to obtain recombinant strains expressing the mutant: BW25113-TPL-C84K, BW25113-TPL-M21R, BW25113-TPL-N2K, BW25113-TPL-D45K, and BW25113-TPL-N372A.

[0050] Table 1 Primers used in this invention

[0051]

[0052] Example 3: Testing of the TPL single mutant of tyrosine phenol lyase

[0053] (1) The five recombinant strains expressing mutants constructed in Example 2 and the recombinant strain expressing wild-type DmTPL (BW25113-DmTPL) as a control were inoculated in LB medium with corresponding resistance and cultured with shaking at 37°C.

[0054] (2) When the bacterial concentration reaches OD600 of 0.6-0.8, add arabinose with a final concentration of 2 g / L for induction, and collect wet bacterial cells by centrifugation after culturing at 30 ℃ for 20 h.

[0055] (3) Wash the bacterial cells with sterile water and centrifuge at 4°C to remove the supernatant. Repeat twice.

[0056] (4) Add lysis buffer (50mM Tris-HCl, 300mM NaCl, pH 8.0) to fully suspend the bacterial cells, break the bacterial cells under low temperature and high pressure, centrifuge at 4℃, and collect the supernatant to obtain crude enzyme solution.

[0057] (5) Take crude enzyme solution to determine the enzyme activity of mutants at different phenol concentrations.

[0058] Composition of enzyme activity assay reaction solution: Tris-HCl 100 mM, sodium pyruvate 50 mM, ammonium chloride 1 M, phenol 50 / 100 / 150 mM, crude enzyme solution (2 g of crude enzyme solution obtained by crushing wet bacterial cells per liter of reaction solution).

[0059] Reaction conditions: 35℃, pH 8.5, reaction time 30 min. After the reaction, the amount of tyrosine produced was determined by HPLC, and the relative enzyme activity was calculated.

[0060] The results are as follows Figure 2 As shown, under low phenol concentration of 50 mM, the enzyme activities of wild-type DmTPL (TPL-WT in the figure) and the five mutants were basically the same. When the phenol concentration increased to 100 mM, the enzyme activity of the wild-type decreased by about half, to about 53.4%, while the mutants TPL-C84K and TPL-M21R showed better phenol tolerance, with their activities remaining above 82% and 90%, respectively. When the phenol concentration was further increased to 150 mM, which is intolerable to most enzymes, the wild-type DmTPL retained only about 20% of its activity, while the mutants TPL-C84K and TPL-M21R still maintained more than 50% of their activity. These results fully demonstrate that when the amino acid at position 84 or 21 is mutated, the tolerance of DmTPL to phenol is improved.

[0061] Example 4: Construction of the TPL double mutant of tyrosine phenol lyase

[0062] (1) To further improve the phenol tolerance of phenol tyrosine phenol lyase, the vector pBAD-TPL-C84K containing the single mutant TPL-C84K constructed in Example 2 was used as a template. The DmTPL gene was subjected to corresponding site superposition mutations using the mutation primers M21R-F / R in Table 1. The PCR amplification products were then used... Dpn After digestion with enzyme I for 2 h, the cells were transformed into E. coli DH5α competent cells. Single clones were selected and sequenced to identify the plasmid pBAD-TPL-C84K-M21R containing the double mutant gene.

[0063] (2) The plasmid containing the double mutant gene was transferred into Escherichia coli BW25113 to obtain the mutant recombinant strain BW25113-TPL-C84K-M21R.

[0064] (3) The mutant recombinant strain BW25113-TPL-C84K-M21R and the control recombinant strain BW25113-DmTPL expressing wild-type DmTPL were respectively inoculated into LB medium with corresponding resistance and cultured at 37°C with shaking.

[0065] (4) When the bacterial concentration reaches OD600 of 0.6-0.8, add arabinose with a final concentration of 2 g / L for induction, and collect wet bacterial cells by centrifugation after culturing at 30℃ for 20 h.

[0066] (5) Resuspend the bacterial cells in sterile water and centrifuge at 4°C to remove the supernatant. Repeat twice.

[0067] (6) Add lysis buffer (50mM Tris-HCl, 300mM NaCl, pH 8.0) to fully suspend the bacterial cells, break the bacterial cells under low temperature and ultra-high pressure, centrifuge at 4℃, and collect the supernatant to obtain crude enzyme solution.

[0068] (7) Take crude enzyme solution to determine the enzyme activity of mutant under different phenol conditions.

[0069] Composition of enzyme activity assay reaction solution: Tris-HCl 100 mM, sodium pyruvate 50 mM, ammonium chloride 1 M, phenol 50 / 100 / 150 mM, crude enzyme solution (2 g of crude enzyme solution obtained by crushing wet bacterial cells per liter of reaction solution).

[0070] Reaction conditions: 35℃, pH 8.5, reaction time 30 min. After the reaction, the amount of tyrosine produced was determined by HPLC and the relative enzyme activity was calculated.

[0071] The results are as follows Figure 3 As shown, when the phenol concentration increased from 50 mM to 100 mM, the enzyme activity of wild-type DmTPL (TPL-WT in the figure) was reduced by about half, while the enzyme activity of the mutant TPL-C84K-M21R was basically unaffected. When the phenol concentration reached 150 mM, wild-type DmTPL retained only about 20% of its activity, while the mutant TPL-C84K-M21R still maintained about 70% of its activity. This result indicates that simultaneous mutations at sites 84 and 21 in DmTPL can further enhance its tolerance to phenol.

[0072] Example 5: Test of catalytic efficiency of tyrosine phenol lyase mutant at high phenol concentration

[0073] (1) The mutant recombinant strain BW25113-TPL-C84K-M21R and the wild-type recombinant strain BW25113-DmTPL as the control were respectively inoculated into LB medium with corresponding resistance and cultured with shaking at 37 °C.

[0074] (2) When the bacterial concentration reaches OD600 of 0.6-0.8, add arabinose with a final concentration of 2 g / L for induction, and collect wet bacterial cells by centrifugation after culturing at 30 ℃ for 20 h.

[0075] (3) Take 240 g of wet bacterial weight and suspend it in 500 mL of deionized water. Perform low temperature and ultra-high pressure cell disruption to obtain wild-type tyrosine phenol lyase DmTPL (control) and tyrosine phenol lyase mutant TPL-C84K-M21R reaction enzyme solution.

[0076] (4) The above-mentioned enzyme solution was added to an enzyme-catalyzed reaction system containing 150 mM phenol to carry out the reaction.

[0077] The enzymatic reaction system consists of: 990 mM sodium pyruvate, 1150 mM ammonium sulfate, 150 mM phenol, 0.12 mM pyridoxal phosphate (PLP), and a reaction enzyme solution obtained by crushing 240 g of wet bacterial cells. The total volume of the enzymatic reaction system is 6 L. During the reaction, 750 g / L phenol is added to ensure that the concentration of phenol in the reaction system does not exceed 150 mM.

[0078] Reaction conditions: 45 ℃, pH maintained at 8.5 using 25% ammonia solution.

[0079] Samples were taken every hour during the reaction, and the concentrations of pyruvate, phenol, and tyrosine were monitored using HPLC.

[0080] according to Figure 4 The results show that under high phenol concentration of 150 mM, the initial reaction rate catalyzed by the mutant TPL-C84K-M21R can reach approximately 400 mM / h, which is 2.25 times that of the wild-type DmTPL (TPL-WT in the figure). As the reaction progresses, the tyrosine production rate catalyzed by the mutant remains more than twice that of the wild-type. After 8 hours of reaction, the tyrosine production rate of the wild-type DmTPL drops to less than 50 mM / h, while the tyrosine production rate of the mutant TPL-C84K-M21R can still reach over 100 mM / h. This result fully demonstrates that the mutant TPL-C84K-M21R has strong phenol tolerance and can maintain a high-speed catalytic level for a long time under high phenol concentration of 150 mM, possessing the potential for large-scale and efficient tyrosine production.

[0081] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A tyrosine phenol lyase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO:1, the mutation was performed at sites selected from the following sites or combinations thereof: 1) The 84th amino acid is mutated from C to K; 2) The 21st amino acid is mutated from M to R.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

4. Use of the tyrosine phenol lyase mutant of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 for the production of tyrosine.

5. A method for producing tyrosine, comprising using pyruvate or its salt, phenol, or ammonium salt as a substrate, contacting the substrate with the tyrosine phenol lyase mutant as described in claim 1, thereby enzymatically synthesizing tyrosine.

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