Tyrosine phenol lyase mutant as well as preparation method and application thereof

Abstract

The invention discloses a tyrosine phenol lyase mutant as well as a preparation method and application thereof, and relates to the field of biology. The tyrosine phenol lyase mutant provided by the invention has the remarkable advantages of high catalytic activity and high phenol tolerance. In practical application, the high activity means that the enzyme dosage can be remarkably reduced under the same catalytic condition, so that the production cost is effectively controlled, and the production efficiency is improved; meanwhile, the excellent phenol tolerance can support a reaction system to adopt higher phenol concentration and faster flow rate, so that the reaction time is shortened, the substrate conversion level and the final titer of the product can be further improved, and key support is provided for large-scale and economical operation of related processes.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to tyrosine phenol lyase mutants, their preparation methods, and applications. Background Technology

[0002] L-Tyrosine, an essential amino acid for protein synthesis in animals, plays a crucial role in the growth, development, and metabolism of humans and animals, and is widely used in various industries such as food, feed, pharmaceuticals, chemicals, and cosmetics. Due to its ability to enhance memory and mental acuity, it is often used as a dietary supplement and appetite suppressant. Simultaneously, it is an important precursor to neurotransmitters such as dopamine and adrenaline, participating in the regulation of brain activity. Furthermore, L-Tyrosine has significant applications in the pharmaceutical field as a precursor for the synthesis of high-value compounds, such as in the preparation of levodopa for treating Parkinson's disease and p-hydroxycinnamic acid in nutritional supplements.

[0003] Currently, the main methods for producing L-tyrosine include hair hydrolysis, fermentation, and enzymatic catalysis. Hair hydrolysis extracts L-tyrosine through hydrolysis, decolorization, and purification of natural hair. However, due to the complex amino acid composition of natural hair, the separation and extraction of high-purity L-tyrosine is difficult, yields are low, and it causes significant environmental pollution; therefore, this method has been gradually phased out. Fermentation utilizes microorganisms to synthesize L-tyrosine de novo using simple carbon sources such as glucose and glycerol. However, it suffers from long fermentation cycles, low acid production efficiency, high production costs, and high extraction process stress, making it difficult to meet the needs of industrial production. Enzymatic catalysis utilizes highly active tyrosine phenol lyase (TPL) to catalyze the synthesis of L-tyrosine from pyruvate, phenol, and ammonium ions in the presence of coenzyme PLP.

[0004] Currently, the activity of TPL enzymes is insufficient to meet market demand. Low-activity TPL enzymes mean that a large amount of bacterial cells need to be used in the production process, and the higher enzyme cost reduces the product's competitiveness. Furthermore, TPL enzymes have poor phenol tolerance, for example, from phenol derived from... Rhodobacter capsulatus The activity of TPL enzyme decreased by more than 90% at phenol concentration of 7.5 g / L (80 mM), originating from... Pantoea agglomerans The activity of TPL enzyme is almost zero at an initial phenol concentration of 11.8 g / L (125 mM). This poor phenol tolerance limits the industrial application of TPL enzyme. For example, the phenol concentration cannot be too high during the catalytic process; real-time monitoring and control of the upper limit are necessary. Even slight errors can lead to (localized) high concentrations of phenol that can fatally damage the enzyme, increasing the complexity of the process. Furthermore, TPL enzyme is prone to inactivation in the later stages of the reaction. To ensure successful catalysis, it is often necessary to increase the enzyme dosage or add fresh enzyme solution later, increasing production costs.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a tyrosine phenol lyase mutant, its preparation method, and its application.

[0007] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a tyrosine phenol lyase mutant having a mutation at any one or more of the following positions in the amino acid sequence of wild-type tyrosine phenol lyase: position 49, position 103, position 186, position 217, and position 385.

[0008] Secondly, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the tyrosine phenol lyase mutant described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid molecules described in the foregoing embodiments or the recombinant vector described in the foregoing embodiments.

[0011] Fifthly, embodiments of the present invention provide a catalyst comprising the tyrosine phenol lyase mutant described in the foregoing embodiments.

[0012] In a sixth aspect, embodiments of the present invention provide a method for preparing the tyrosine phenol lyase mutant as described in the foregoing embodiments, comprising: artificially synthesizing the tyrosine phenol lyase mutant or culturing the recombinant cells described in the foregoing embodiments.

[0013] In a seventh aspect, embodiments of the present invention provide the use of the tyrosine phenol lyase mutant as described in the foregoing embodiments, or the isolated nucleic acid molecule as described in the foregoing embodiments, or the recombinant vector as described in the foregoing embodiments, or the recombinant cell as described in the foregoing embodiments, or the catalyst as described in the foregoing embodiments, in the preparation of L-tyrosine.

[0014] Eighthly, embodiments of the present invention provide a method for preparing L-tyrosine, comprising the following steps: adding the tyrosine phenol lyase mutant described in the foregoing embodiments, or the recombinant cells described in the foregoing embodiments, or the catalyst described in the foregoing embodiments to a reaction system containing serine and phenol to carry out a catalytic reaction.

[0015] The present invention has the following beneficial effects: The tyrosine phenol lyase mutant provided by this invention possesses the significant advantages of both high catalytic activity and high phenol tolerance. In practical applications, its high activity means that the amount of enzyme used can be significantly reduced under the same catalytic conditions, thereby effectively controlling production costs and improving production efficiency. At the same time, its excellent phenol tolerance allows for the use of higher phenol concentrations and faster flow rates in the reaction system. This not only helps to shorten the reaction time but also further improves the substrate conversion level and the final product potency, providing key support for the large-scale and economical operation of related processes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a synthetic route for the production of L-tyrosine using tyrosine phenol lyase catalysis. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0020] On one hand, embodiments of the present invention provide a tyrosine phenol lyase mutant having mutations at any one or more of the following positions in the amino acid sequence of wild-type tyrosine phenol lyase: position 49, position 103, position 186, position 217, and position 385.

[0021] In an optional embodiment, the amino acid sequence of the wild-type tyrosine phenol lyase has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0022] In an optional embodiment, the mutation at position 49 is T49S.

[0023] In an optional embodiment, the mutation at position 103 is: E103K, E103Y, or E103R.

[0024] In an optional embodiment, the mutation at position 186 is either L186C or L186T.

[0025] In an optional embodiment, the mutation at position 217 is: R217D.

[0026] In an optional embodiment, the mutation at position 385 is either S385T or S385A.

[0027] In an optional embodiment, the tyrosine phenol lyase mutant has any of the following mutation combinations relative to the wild-type tyrosine phenol lyase: T49S / E103Y / R217D, T49S / E103R / S385T, T49S / E103Y / R217D / S385A, E103Y / L186C / S385T, and T49S / E103R / L186C / R217D / S385T.

[0028] In an optional embodiment, the at least 80% includes 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0029] On the other hand, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the tyrosine phenol lyase mutant described in any of the foregoing embodiments.

[0030] On the other hand, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in any of the foregoing embodiments.

[0031] In optional embodiments, the recombinant vector is an expression vector or a cloning vector, optionally an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells via transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0032] On the other hand, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid molecules or the recombinant vectors described in any of the foregoing embodiments.

[0033] In optional embodiments, the recombinant cells can be prokaryotic cells, eukaryotic cells, or bacteriophages. The aforementioned prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*; insect cells such as *Ardisia crenata*; mammalian cells such as BHK cells, CHO cells, COS cells, NSO cells, 293 series cells, HepG2, HEK293 cell lines, Huh7 cells, and myeloma cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0034] In an optional embodiment, the recombinant cells comprise recombinant bacteria. The recombinant bacteria can be prokaryotic or eukaryotic recombinant bacteria. The prokaryotic recombinant bacteria include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The eukaryotic recombinant bacteria include, but are not limited to, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharizoa*, *Trichoderma*, and other fungi.

[0035] On the other hand, embodiments of the present invention provide a catalyst containing the tyrosine phenol lyase mutant described in any of the foregoing embodiments; In an optional embodiment, the catalyst comprises: recombinant cells or cultures thereof as described in any of the foregoing embodiments.

[0036] In an optional embodiment, the preparation of the recombinant cell culture includes: S1: culturing the recombinant cells to OD. 600 S2: Add isopropyl-β-D-thiogalactoside (IPTG) to a concentration of 0.01-1 mM, and incubate at 20-30℃ for 20-30 h; S3: Centrifuge, discard the supernatant, and collect the precipitate.

[0037] In an optional embodiment, the culture temperature in step S1 can be 30~45℃, or optionally 36~38℃.

[0038] In an optional embodiment, in step S2, the concentration of IPTG is 0.05~0.5mM, optionally 0.05~0.2mM.

[0039] On the other hand, embodiments of the present invention provide a method for preparing the tyrosine phenol lyase mutant as described in any of the foregoing embodiments, which includes: artificially synthesizing the tyrosine phenol lyase mutant or culturing the recombinant cells described in any of the foregoing embodiments.

[0040] On the other hand, embodiments of the present invention provide the use of the tyrosine phenol lyase mutant as described in any of the foregoing embodiments, or the isolated nucleic acid molecule as described in any of the foregoing embodiments, or the recombinant vector as described in any of the foregoing embodiments, or the recombinant cell as described in any of the foregoing embodiments, or the catalyst as described in any of the foregoing embodiments, in the preparation of L-tyrosine.

[0041] Furthermore, embodiments of the present invention provide a method for preparing L-tyrosine, which includes the following steps: adding the tyrosine phenol lyase mutant described in any of the foregoing embodiments, the recombinant cells described in any of the foregoing embodiments, or the catalyst described in any of the foregoing embodiments to a reaction system containing serine and phenol to carry out a catalytic reaction.

[0042] In an optional embodiment, the temperature of the catalytic reaction is 25~45°C, and the pH is 7~8; the reaction temperature can be any one or any two of 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, and 45°C; the pH can be any one or any two of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.

[0043] In an optional embodiment, the concentration of serine in the reaction system is 60-80 g / L, the concentration of phenol is 6-10 g / L, and the concentration of the tyrosine phenol lyase mutant, the recombinant cell, or the culture of the recombinant cell is 10-20 g / L.

[0044] In an optional embodiment, the concentration of serine can be any one or any two of 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 g / L.

[0045] In an optional embodiment, the concentration of the tyrosine lyase mutant or the recombinant cell or the culture of the recombinant cell (e.g., wet cells expressing the tyrosine lyase mutant) can be any one or any two of 10, 12, 14, 16, 18 and 20 g / L.

[0046] In an optional embodiment, serine deaminase or bacterial cells expressing serine deaminase, surfactant, and coenzyme PLP are also added to the reaction system.

[0047] Specifically, PLP, pyridoxal phosphate, is known in English as Pyridoxal 5'-phosphate.

[0048] The preparation method provided in the embodiments of the present invention is a dual-enzyme catalytic system using serine and phenol as raw materials: The first step involves the conversion of serine to pyruvate and ammonia via serine deaminase; the second step utilizes tyrosine phenol lyase (mutant) to further catalyze the intermediate products pyruvate and ammonia with the substrate phenol to generate L-tyrosine, which can be referred to... Figure 1 .

[0049] In an optional embodiment, the surfactant is Triton.

[0050] In an optional embodiment, in the reaction system, the concentration of the serine deaminase or the bacterial cells expressing the serine deaminase is 10-20 g / L, the concentration of Triton is 0.1-2 g / L, and the concentration of the coenzyme PLP is 0.01-1 g / L.

[0051] In an optional embodiment, the concentration of the serine deaminase or the bacterial cells (wet cells) expressing the serine deaminase can be any one or any two of 10, 12, 14, 16, 18 and 20 g / L.

[0052] In an optional embodiment, the concentration of the triton may be any one or any two of 0.1, 0.5, 1, 1.5, 2 g / L.

[0053] In an optional embodiment, the concentration of the coenzyme PLP can be any one or any two of 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, 0.8 and 1 g / L.

[0054] In an optional embodiment, the preparation method further includes adding a 40%–60% (w / w) phenol solution after the catalytic reaction has been carried out for 30–90 min, and then continuing the reaction for another 9–15 h. Specifically, the 30–90 min can be 50–70 min, the 40%–60% can be 45%–55%, and the 9–15 h can be any one or any combination of 9, 10, 11, 12, 13, 14, and 15 h.

[0055] In an optional embodiment, the flow rate of the phenol solution is 12-16 g / h, specifically any one or any two of 12, 13, 14, 15 and 16 g / h.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1: Construction of a strain for producing tyrosine phenol lyase (Cf-TPL) Selected from Citrobacter freundiiThe amino acid sequence of ATCC8090 tyrosine phenol lyase (Cf-TPL) (NCBI accession number: WP_003837154.1, amino acid sequence as shown in SEQ ID NO:1) was chemically synthesized after codon optimization (nucleotide sequence as shown in SEQ ID NO:2), and ligated into the plasmid vector pET-28a(+) through NcoⅠ and XhoⅠ restriction sites. The recombinant enzyme expression strain was constructed by introducing it into the host strain Escherichia coli BL21(DE3) using CaCl2-mediated chemical transformation.

[0058] Example 2: Saturation Mutation Modification to Enhance Cf-TTPL Enzyme Activity and Phenol Tolerance Using Cf-TPL (amino acid sequence as shown in SEQ ID NO:1) as the wild type, saturated mutant libraries were constructed based on the degenerate codon NNK at the following sites: F36, L78, T49, S51, Q98, R100, E103, Y122, F123, T124, T125, T126, N185, L186, R217, K256, K257, M379, E380, R381, S385, A386, R404, L446, F448, and F449.

[0059] Construction of saturated mutant library: Using pET-28a(+)-Cf-TPL plasmid DNA as a template, PCR amplification was performed using PrimeStarMax enzyme. The PCR reaction system was 50 μL: 25 μL of PrimeStarMax enzyme, 2 μL each of upstream and downstream primers containing degenerate bases, 1 μL of template DNA, and the remainder was made up with pure water. The reaction program was: 98℃ pre-denaturation for 5 min; followed by 98℃ denaturation for 30 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min 10 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. A fragment product of approximately 6000 bp was obtained, digested with DpnI enzyme, transformed into E. coli BL21(DE3) competent cells, plated, and incubated overnight at 37℃. Single colonies were picked and transferred to 96-well plates to obtain the saturated mutant library.

[0060] 96-well plate culture: Clones were picked from the saturated mutant library and transferred to 96-well plates. 200 μL of LB liquid medium containing 50 mg / L kanamycin was added to each well, and the plates were incubated overnight at 37°C and 250 rpm to obtain primary seed culture. 40 μL of the primary seed culture was transferred to TB liquid medium containing 50 mg / L kanamycin and incubated at 37°C until OD500. 600 When the concentration of the culture medium is 0.6-0.8, add IPTG at a final concentration of 0.1 mM for induction, and incubate overnight at 25°C to obtain the fermentation broth.

[0061] High-throughput screening: Transfer 50 μL of fermentation broth from a 96-well plate to a new plate and divide it into two groups: one group was treated with 50 μL of pure water per well to determine the basic enzyme activity of the mutant strain; the other group was treated with 50 μL of an aqueous solution containing 2.4 wt% phenol (pH 7.5) per well, and incubated at 37°C with shaking for 8 hours to determine the residual enzyme activity after phenol treatment. Reaction solution preparation: Weigh 1.04 g of sodium pyruvate, 0.85 g of phenol, 1.18 g of ammonium sulfate, 0.1 g of Triton, and 0.02 g of PLP, dissolve them in pure water, adjust the pH to 7.5, and bring the volume to 80 mL. Add 400 μL of the reaction solution to each well of the plate containing the bacteria, incubate at 37°C with shaking for 1 hour, and then terminate the reaction by adding 50 μL of 0.6M HCl. The colorimetric system was freshly prepared by mixing 5 g / L salicylaldehyde solution and 75 g / L NaOH solution at a 1:1 volume ratio. 160 μL of the colorimetric solution was added to the ELISA plate, followed by 40 μL of the stop reaction solution. The plate was incubated at room temperature for 30 min, and the absorbance at 465 nm was measured using an ELISA reader. The principle is that salicylaldehyde forms a colorimetric complex with pyruvate under alkaline conditions. The residual pyruvate content is quantified by the 465 nm absorbance: the lower the absorbance, the more pyruvate is consumed, and the higher the enzyme activity of the corresponding mutant strain. The enzyme activity grading criteria were based on the starting strain control: a 10-20% decrease in absorbance was defined as "+", 20-40% as "++", 40-60% as "+++", and above 60% as "++++".

[0062] Sequencing was performed on the enzyme activity-enhancing strains to determine the mutation types. The sequencing results showed that mutant strains T49S, E103K, E103Y, E103R, L186C, L186T, R217D, and S385T exhibited varying degrees of enhancement in enzyme activity and phenol tolerance. Among them, mutant strain R217D showed the most significant enhancement in both enzyme activity and phenol tolerance.

[0063] Table 1 Results

[0064] Example 3: Randomized combination mutation modification to enhance Cf-TTPL enzyme activity and phenol tolerance Beneficial mutant strains were combined using staggered extension PCR (StEP). The unit enzyme activity of the wet mutant cells and the relative enzyme activity after phenol treatment were determined by shake-flask fermentation. The specific procedure is as follows: Beneficial mutant strains were taken from preservation tubes and inoculated into 5 mL LB medium containing 50 mg / L kanamycin at a 1% inoculum rate. The culture was incubated at 37°C and 200 rpm for 15 h to obtain primary seed culture. Subsequently, the primary seed culture was transferred to 50 mL TB medium containing 50 mg / L kanamycin at a 4% inoculum rate and incubated at 37°C and 220 rpm until the bacterial OD reached the target value. 600When the expression level reaches 0.6-0.8, IPTG at a final concentration of 0.1 mM is added to induce expression. After induction at 25℃ and 220 rpm for 24 h, the supernatant is removed by centrifugation at 4000 rpm for 15 min, and the wet cells are collected.

[0065] The enzyme activity assay system consisted of 5 g / L wet bacterial cells containing 10.4 g / L sodium pyruvate, 8.5 g / L phenol, 1.18 g / L ammonium sulfate, 1 g / L Triton, and 0.2 g / L PLP. The pH was adjusted to 7.5, and the enzyme activity of the mutant strain was measured after reacting for 30 min. Separately, bacterial cells were treated in 1.2 wt% phenol solution (pH 7.5) for 8 h, and their residual enzyme activity was measured.

[0066] Enzyme activity related definitions: Under the above reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol L-tyrosine per minute is defined as 1 enzyme activity unit (U); unit enzyme activity refers to the enzyme activity units (U / g) contained in each gram of wet cells; relative enzyme activity is calculated based on the unit enzyme activity of wild-type Cf-TPL (100%).

[0067] After verification and screening, five combined mutant strains with the most significant improvement in activity and phenol tolerance were obtained (see Table 2).

[0068] Table 2 Results

[0069] Among them, the best mutant strain was Cf-TPL mutant 5 (T49S / E103R / L186C / R217D / S385T), which not only increased the activity by 80%, but also greatly improved the phenol tolerance. After treatment in 1.2wt% phenol solution (pH 7.5) for 8 hours, the enzyme activity remained at more than 93%.

[0070] Example 4: Fermentation to obtain serine deaminase cells The serine deaminase gene (NCBI accession number: WP_108118731.1) was amplified from the genome of Escherichia coli MG1655, cloned into the pET-28a(+) plasmid, introduced into Escherichia coli BL21(DE3), and fermented for expression according to Example 3 to obtain serine deaminase cells.

[0071] Example 5: Preparation of tyrosine catalyzed by Cf-TPL strain Catalysis was carried out in a 3L reactor with a final volume of 1L. In different experimental groups, 60-80g of serine, 5g of phenol, 1g of Triton, and 0.1g of PLP were added sequentially. After dissolving in pure water, the pH of the system was adjusted to 7.5. 10g of serine deaminase cells and 10-20g of Cf-TPL wild-type cells were added, and the reaction was carried out at 37℃ and 300rpm, maintaining the pH of the system at 7.5 during the process. After 1h of reaction, 50% (w / w) phenol solution was added at a flow rate of 12g / h. The reaction was terminated after 9-15h from the start of serine addition.

[0072] Sampling was performed to detect residual serine and phenol, as well as the potency of the target product L-tyrosine, and the yield was calculated. The catalyst solution sample was diluted in one step with 1M HCl and then in two steps with 10mM sodium dihydrogen phosphate solution before being filtered through a membrane for analysis.

[0073] Tyrosine and phenol were detected by HPLC using an Agilent XDB-C18 column (150×4.6 mm, 5µm), a column temperature of 40℃, a mobile phase of methanol:0.1% phosphoric acid water = 10:90 (V / V), a flow rate of 1.0 mL / min, a UV detector wavelength of 275 nm, and a run time of 15 min. Characteristic peaks for tyrosine and phenol appeared at approximately 2.84 min and 12.18 min, respectively.

[0074] Serine was detected by HPLC using an Agilent C18 column (150 × 4.6 mm, 4 µm), a column temperature of 35 °C, a mobile phase of 5 mmol / L sodium dodecyl sulfate (pH 2.7): acetonitrile = 80:20 (V / V), a flow rate of 1.0 mL / min, a UV detector of 192 nm, and a run time of 20 min. Serine showed a characteristic peak at approximately 8.50 min.

[0075] In the reaction catalyzed by wild-type TPL enzyme, when the final concentration of wild-type TPL enzyme cells was 20 g / L and the final concentration of serine was 60 g / L, the final titer of the tyrosine product reached 93.5 g / L, and the reaction yield was 90.34%. When the amount of wild-type TPL enzyme cells was gradually decreased from 20 g / L to 10 g / L, or when the concentration of serine was gradually increased from 60 g / L to 80 g / L, the product titer and yield decreased to varying degrees.

[0076] Table 3 Results

[0077] Example 6: Preparation of tyrosine catalyzed by Cf-TPL mutant strain 4 Catalysis was carried out in a 3L reactor with a final volume of 1L. In different experimental groups, 60-80g of serine, 5-10g of phenol, 1g of Triton, and 0.1g of PLP were added sequentially. After dissolving in pure water, the pH of the system was adjusted to 7.5. 10g of serine deaminase cells and 10-20g of Cf-TPL mutant 4 cells were added, and the reaction was carried out at 37℃ and 300rpm, maintaining the pH of the system at 7.5 during the process. After 1h of reaction, 50% (w / w) phenol solution was added at a flow rate of 12g / h. The reaction was terminated after 9-15h from the start of serine addition.

[0078] Sampling was performed to detect residual serine and phenol levels, as well as the titer of the target product L-tyrosine, and the yield was calculated. In the reaction catalyzed by Cf-TPL mutant strain 4, at a final cell concentration of 20 g / L and a final serine concentration of 80 g / L, the final titer of the tyrosine product reached 125.2 g / L, with a reaction yield of 90.8%, demonstrating superior catalytic performance compared to the wild-type Cf-TPL strain.

[0079] Increasing the concentration and flow rate of phenol in the substrate can shorten the reaction time from 15 hours to 9 hours without significantly changing the product titer and yield. However, further reducing the bacterial cell concentration from 20 g / L to 10 g / L results in varying degrees of decrease in both product titer and yield.

[0080] Table 4 Results

[0081] Example 7: Preparation of tyrosine catalyzed by Cf-TPL mutant strain 5 Catalysis was carried out in a 3L reactor with a final volume of 1L. In different experimental groups, 60-80g of serine, 5-10g of phenol, 1g of Triton, and 0.1g of PLP were added sequentially. After dissolving in pure water, the pH of the system was adjusted to 7.5. 10g of serine deaminase cells and 10-20g of Cf-TPL mutant cells were added, and the reaction was carried out at 37℃ and 300rpm, maintaining the pH of the system at 7.5 during the process. After 1h of reaction, 50% (w / w) phenol solution was added at a flow rate of 12g / h. The reaction was terminated after 9-15h from the start of serine addition.

[0082] Sampling was performed to detect residual serine and phenol levels, as well as the titer of the target product L-tyrosine, and the yield was calculated. In the reaction catalyzed by Cf-TPL mutant strain 5, at a final cell concentration of 20 g / L and a final serine concentration of 80 g / L, the final titer of the tyrosine product reached 126.5 g / L, with a reaction yield of 91.7%. This catalytic effect was superior to that of the wild-type Cf-TPL strain and mutant strain 4.

[0083] Increasing the concentration and flow rate of phenol in the substrate can shorten the reaction time from 15 hours to 9 hours, resulting in a slight increase in product potency and yield. Further, gradually reducing the bacterial cell concentration from 20 g / L to 10 g / L further slightly improves product potency and yield. This is mainly because mutant strain 5 has higher enzyme activity and phenol tolerance, enabling it to efficiently convert the substrate into the product with lower bacterial cell concentrations and higher substrate and phenol concentrations. Furthermore, the shortened catalytic time and reduced bacterial cell concentration lead to lower levels of chemical and biodegradation of the intermediate pyruvate, thus increasing the product yield.

[0084] Table 5 Results

[0085] The sequence information involved in this application is shown in the table below.

[0086] Table 4 Sequence

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tyrosine phenol lyase mutant, characterized in that, It has mutations at any one or more sites in the amino acid sequence of wild-type tyrosine phenol lyase, at positions 49, 103, 186, 217, and 385. Optionally, the amino acid sequence of the wild-type tyrosine phenol lyase has at least 80% identity with the sequence shown in SEQ ID NO:

1.

2. The mutant according to claim 1, characterized in that, The mutation at position 49 is T49S; And / or, the mutation at position 103 is: E103K, E103Y, or E103R; And / or, the mutation at position 186 is: L186C or L186T; And / or, the mutation at position 217 is: R217D; And / or, the mutation at position 385 is: S385T or S385A.

3. The mutant according to claim 2, characterized in that, The tyrosine phenol lyase mutant has any of the following mutation combinations relative to the wild-type tyrosine phenol lyase: T49S / E103Y / R217D, T49S / E103R / S385T, T49S / E103Y / R217D / S385A, E103Y / L186C / S385T, and T49S / E103R / L186C / R217D / S385T.

4. An isolated nucleic acid molecule, characterized in that, It encodes the tyrosine phenol lyase mutant according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that, It contains the isolated nucleic acid molecules as described in claim 4.

6. A recombinant cell, characterized in that, It contains the isolated nucleic acid molecule as described in claim 4 or the recombinant vector as described in claim 5; Optionally, the recombinant cells include recombinant bacteria.

7. A catalyst, characterized in that, It contains the tyrosine phenol lyase mutant according to any one of claims 1 to 3; Optionally, the catalyst comprises: the recombinant cells or cultures thereof as described in claim 6.

8. The method for preparing the tyrosine phenol lyase mutant according to any one of claims 1 to 3, characterized in that, It includes: The tyrosine phenol lyase mutant is artificially synthesized or the recombinant cells of claim 6 are cultured.

9. The use of the tyrosine phenol lyase mutant according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6, or the catalyst according to claim 7 in the preparation of L-tyrosine.

10. A method for preparing L-tyrosine, characterized in that, It includes the following steps: In a reaction system containing serine and phenol, the tyrosine phenol lyase mutant of any one of claims 1 to 3, the recombinant cell of claim 6, or the catalyst of claim 7 is added to carry out the catalytic reaction. Optionally, the temperature of the catalytic reaction is 25~45℃, and the pH is 7~8; Optionally, in the reaction system, the concentration of serine is 60-80 g / L, the concentration of phenol is 6-10 g / L, and the concentration of the tyrosine phenol lyase mutant, the recombinant cell, or the culture of the recombinant cell is 10-20 g / L. Optionally, serine deaminase or bacterial cells expressing serine deaminase, surfactant, and coenzyme PLP are also added to the reaction system. Optionally, the surfactant is Triton; Optionally, in the reaction system, the concentration of the serine deaminase or the bacterial cells expressing the serine deaminase is 10-20 g / L, the concentration of Triton is 0.1-2 g / L, and the concentration of the coenzyme PLP is 0.01-1 g / L. Optionally, the preparation method further includes adding a 40% to 60% (w / w) phenol solution after the catalytic reaction has been carried out for 30 to 90 minutes, and then continuing the reaction for another 9 to 15 hours; Optionally, the flow rate of the phenol solution is 12~16 g / h.

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