Tyrosine phenol lyase mutant and application thereof in production of L-tyrosine

Abstract

The invention discloses a tyrosine phenol lyase mutant and application thereof in production of L-tyrosine, and belongs to the technical field of bioengineering. According to the tyrosine phenol lyase mutant, amino acid mutation occurs on an amino acid sequence as shown in SEQ ID NO.3, and mutation sites comprise at least one of the 51st site, the 70th site, the 72nd site, the 260th site and the 299th site. The mutant has relatively high catalytic activity and has phenol tolerance. When the mutant is used for preparing the L-tyrosine, the yield and the preparation efficiency of the L-tyrosine can be remarkably improved, and the mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a tyrosine phenol lyase mutant and its application in the production of L-tyrosine. Background Technology

[0003] Currently, the main methods for preparing L-tyrosine include extraction, chemical synthesis, and biotransformation. Extraction primarily uses natural protein resources as raw materials, extracting L-tyrosine through hydrolysis, decolorization, and crystallization. However, the high cost and low yield of this method limit its industrial application. Chemical synthesis involves the hydroxylation of L-phenylalanine or the condensation of p-hydroxybenzaldehyde with hydantoin, alkaline hydrolysis, and transamination. This method is complex, energy-intensive, and generates significant pollutants, and has been gradually phased out. Biotransformation, with its advantages of high specificity, environmental friendliness, mild reaction conditions, and elimination of the need for multi-step separation and purification, has attracted widespread attention.

[0004] Biotransformation methods are divided into microbial fermentation and enzymatic catalysis. Microbial fermentation involves complex process control, requiring conditions that are suitable for both microbial growth and the accumulation of metabolites. It has a long production cycle, low efficiency, and is unsuitable for industrial applications. Enzymatic catalysis offers various routes for L-tyrosine preparation, but the most studied is the tyrosine phenol lyase-mediated reaction. Sha Xu et al. screened a reaction originating from microorganisms... Erwinia herbicola The tyrosine phenol lyase (TPL) was used to convert pyruvate, ammonia, and phenol into L-tyrosine, ultimately yielding 48.5 g / L of L-tyrosine. Although the enzymatic method is more efficient and gentler in comparison, its efficiency is still relatively low, and the production cost of L-tyrosine is relatively high.

[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 and its application in the production of L-tyrosine. By performing site-directed mutagenesis on tyrosine phenol lyase, the obtained tyrosine phenol lyase mutant can improve catalytic activity and phenol tolerance, and significantly increase the yield and production efficiency of L-tyrosine.

[0007] This invention is implemented as follows: Existing routes for preparing L-tyrosine include Figure 1 As shown: L-tyrosine is prepared by conversion of phenol and pyruvate as substrates via wild-type tyrosine phenol lyase (TPL). To improve the catalytic activity of TPL and its tolerance to the substrate phenol, the following optimizations were made in this invention: For wild-type TPL, this invention first screens for TPLs with higher enzyme activity from different sources (derived from...).Vibrio mimicus The TPL mutant can be obtained by performing site-directed mutagenesis on the selected TPL (Genbank ID KFE31745.1, amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4). Specifically, the mutation sites of the TPL mutant include at least one of positions 51, 70, 72, 260, and 299.

[0008] The mutation at position 51 is as follows: aspartic acid is mutated to any one of asparagine, threonine, or glutamine, namely D51N, D51T, and D51Q.

[0009] The mutation at position 70 is as follows: glutamic acid is mutated to any one of valine, alanine, methionine, serine, or aspartic acid, namely E70V, E70A, E70M, E70S, and E70D.

[0010] The mutation at position 72 is as follows: tyrosine is mutated to any one of histidine, proline, or phenylalanine, namely Y72H, Y72P, and Y72F.

[0011] The mutation at position 260 is as follows: lysine is mutated to any one of leucine, valine, or methionine, namely K260L, K26V, and K260M.

[0012] The mutation at position 299 is as follows: threonine is mutated to any one of lysine, phenylalanine, or tyrosine, namely T299K, T299F, and T299Y.

[0013] The mutations at positions 50, 70, 72, 260, and 299 can improve the catalytic activity and phenol tolerance of TPL, thereby enabling the efficient preparation of L-tyrosine.

[0014] Furthermore, the mutation modes of the TPL mutant include, but are not limited to, D51N, D51T, D51Q, D51T / E70V, D51T / E70A, D51T / E70M, D51T / E70S, D51T / E70D, D51T / E70M / Y72H, D51T / E70M / Y72P, D51T / E70M / Y72F, and D51T / E 70M / Y72H / K260L, D51T / E70M / Y72H / K260V, D51T / E70M / Y72H / K260M, D51T / E70M / Y7 2H / K260V / T299K, D51T / E70M / Y72H / K260V / T299F, D51T / E70M / Y72H / K260V / T299Y.

[0015] Furthermore, among the many mutation modes, the mutant corresponding to D51T / E70M / Y72H / K260V / T299K has higher enzyme activity and better L-tyrosine production efficiency. Its amino acid sequence is shown in SEQ ID NO.7 and its nucleotide sequence is shown in SEQ ID NO.8.

[0016] The aforementioned TPL mutant can be used to obtain related biological materials, including: nucleic acid molecules encoding the aforementioned TPL mutant; expression cassettes containing the aforementioned nucleic acid molecules; recombinant vectors containing the aforementioned nucleic acid molecules or recombinant vectors containing the aforementioned expression cassettes; recombinant bacteria containing the aforementioned nucleic acid molecules or recombinant bacteria containing the aforementioned expression cassettes or recombinant bacteria containing the aforementioned recombinant vectors.

[0017] In some embodiments, the method for constructing recombinant bacteria expressing the above-mentioned TPL mutant includes: ligating the nucleotide sequence of the TPL mutant to an expression vector, transforming the obtained recombinant vector into host cells, culturing the host cells, and inducing the expression of the TPL mutant.

[0018] The expression vector and host cell can be conventional choices in the art; in some embodiments, the expression vector is pET28a(+) and the host cell is Escherichia coli. E.coli BL21(DE3). By using the above method to induce protein expression and obtain crude enzyme solution through cell disruption, the catalytic activity of the obtained TPL mutant was superior to that of the wild-type enzyme.

[0019] Adding the above-mentioned TPL mutant to a reaction system containing phenol can be used to synthesize L-tyrosine. Based on this, the present invention also provides a whole-cell catalyst, including the above-mentioned recombinant bacteria.

[0020] Meanwhile, the present invention also provides a method for producing L-tyrosine, comprising: cleaving recombinant bacteria expressing the above-mentioned TPL mutant, and then adding its supernatant to a transformation system for reaction to obtain L-tyrosine.

[0021] In some embodiments, the host expressing the TPL mutant includes Escherichia coli.

[0022] In some embodiments, the transformation system includes: phenol, sodium pyruvate, ammonium acetate, pyridoxal phosphate, phosphate buffer, and recombinant bacterial cells.

[0023] In some embodiments, the phosphate buffer is a sodium phosphate buffer or a potassium phosphate buffer, or a combination of sodium phosphate buffer and potassium phosphate buffer.

[0024] In some embodiments, the phosphate buffer is selected from at least one of K2HPO4-KH2PO4 buffer and Na2HPO4-NaH2PO4 buffer.

[0025] In some embodiments, the transformation system comprises: 2-20 g / L phenol, 3-30 g / L sodium pyruvate, 2-20 g / L ammonium acetate, 0.02-0.2 g / L pyridoxal phosphate, 20-150 mM phosphate buffer, and 2-10 g / L recombinant bacterial cells.

[0026] In some embodiments, the reaction conditions are: pH 7.5-8.5, temperature 25-35°C, shaking speed 150-250 rpm, and reaction time 1-5 h.

[0027] The present invention has the following beneficial effects: This invention obtained a tyrosine phenol lyase mutant through site-directed mutagenesis. This mutant exhibits high catalytic activity and phenol tolerance. Using this mutant to prepare L-tyrosine, 152.3 g / L of L-tyrosine can be obtained within 2 hours, with a conversion rate of 98.9% and a space-time yield of 76.1 g / L / h. This is superior to the conversion effect of the original enzyme and other mutant enzymes, demonstrating good prospects for industrial application. Attached Figure Description

[0028] 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.

[0029] Figure 1 This is the synthetic route for L-tyrosine in this invention. Detailed Implementation

[0030] 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.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] This invention uses high-performance liquid chromatography (HPLC) to analyze substrate and product concentrations. The specific analytical method is as follows: The conversion solution was analyzed using a Shimadzu 2030C high-performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: mobile phase: methanol:water (1:1), Inertsustain C18 column (4.6×250 mm, 5 μm), flow rate: 1 mL / min, column temperature: 30℃, injection volume: 20 μL, and detection wavelength: 275 nm.

[0033] Example 1 This example demonstrates enzyme screening. 1. Enzyme source and construction of recombinant bacteria Tyrosine phenol lyase was obtained from the NCBI database, derived from... Pasteurella multocida (Genbank ID AIN48037.1) Vibrio mimicus (Genbank number KFE31745.1) and Citrobacter intermedius (Genbank accession number BAA00763.1), and named PmTPL, VmTPL, and CiTPL respectively, three nucleotide sequences were synthesized using a total synthesis method based on the amino acid sequence and codon optimization according to the codon preference of *E. coli*, as shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6 respectively, through conventional genetic engineering operations. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5 respectively. A 6×His-tag was added to the end of the nucleotide sequence, and NdeI and XhoI restriction sites were added to both ends. The gene was cloned into the NdeI and XhoI sites corresponding to pET28a(+) to obtain recombinant expression plasmids pET28a-PmTPL, pET28a-VmTPL, and pET28a-CiTPL. These three plasmids were then transformed into *E. coli*. Escherichia coli Recombinant bacteria were obtained from BL21(DE3) competent cells. Escherichia coli BL21(DE3) / pET28a-PmTPL, Escherichia coli BL21(DE3) / pET28a-VmTPL and Escherichia coli BL21(DE3) / pET28a-CiTPL.

[0034] 2. Induced expression of tyrosine phenol lyases from different sources Recombinant *E. coli* was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain seed culture. The seed culture was then inoculated into fresh LB medium at a 2% inoculation rate and cultured at 37°C and 200 rpm until the bacterial concentration (OD600 nm) reached 0.7. 0.5 mM IPTG was added, and the culture was induced at 28°C for 15 h. The culture was then centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the wet bacterial cells were washed twice with 0.9% physiological saline, centrifuged again, and set aside for later use.

[0035] 3. Comparison of tyrosine phenol lyase activity and phenol tolerance in recombinant bacteria Enzyme activity assay: Wet bacterial cells were sonicated. 1 g of the prepared wet bacterial cells were resuspended in 50 mL of 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 8.0) and sonicated for 15 min at 35 W to obtain a sonicated suspension. The suspension was centrifuged, and the supernatant was collected. 1 mL of the supernatant was used for the reaction. Reaction system: 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 8.0), 4 g / L phenol, 6 g / L sodium pyruvate, 4 g / L ammonium acetate, 0.1 g / L pyridoxal phosphate, and 100 μL TPL supernatant, totaling 1 mL. Phenol tolerance assay: The phenol concentration was increased to 10 g / L, while other conditions remained unchanged, and the change in enzyme activity was measured. Reaction conditions: The reaction was carried out at 30℃ for 5 min. Samples were taken, and the yield of L-tyrosine was determined by HPLC.

[0036] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of L-tyrosine per minute at 30°C and pH 8.0.

[0037] Table 1 Comparison of the activities of each recombinase and phenol tolerance

[0038] Example 2 This example demonstrates the construction and screening of VmTPL unit point mutants. 1. Construction of mutants Site-directed mutagenesis primers were designed based on the parental sequence of VmTPL (amino acid sequence SEQ ID NO.3, nucleotide sequence SEQ ID NO.4). Using rapid PCR technology, a single mutation was introduced at position 51 using recombinant pET28a-VmTPL as a template. The primers are as follows: Forward primer 51D: TCGGACNNAGCGGCACCTCTGCACTGTCTG- SEQ ID NO.9; Reverse primer 51D: TGCCGCTNNNGGTCAGAAAATCGATGTACACGTC- SEQ ID NO.10.

[0039] PCR reaction system: 25 μL of 2×FastPfu Fly Reaction Mix, 2 μL of forward primer 51D (10 μM), 2 μL of reverse primer 51D (10 μM), 1 μL of template DNA, 1 μL of FastPfu Fly DNA Polymerase, and ddH2O added to a final volume of 50 μL.

[0040] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 2 min) 30 cycles; 72℃ for 10 min.

[0041] 2. Mutant Transformation Expression PCR results were verified by agarose gel electrophoresis. The PCR product was digested with DpnI enzyme at 37°C for 1 h and then inactivated at 65°C for 1 min. 10 μL of the PCR product was then added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37℃ and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37℃ for 12 h.

[0042] 3. High-throughput screening of positive transformants Reaction mixture: 50 mM K2HPO4-KH2PO4 buffer (pH 8.0), 4 g / L phenol, 6 g / L sodium pyruvate, 4 g / L ammonium acetate, 0.1 g / L pyridoxal phosphate, and 100 μL TPL lysate supernatant (for phenol tolerance testing, the phenol concentration in the system needs to be increased to 10 g / L); Add 200 μL of LB medium containing a final concentration of 50 mg / L kanamycin to each well of a 96-well plate, pick different single colonies into the 96-well plate, and incubate at 37℃ and 200 rpm until the OD600 reaches 0.5-0.6. Add IPTG to the culture medium to a final concentration of 0.5 mM, induce expression at 28℃ for 12 h, centrifuge at 20℃ and 4200 rpm for 10 min, and discard the supernatant. Add 100 μL of the reaction mixture to a 96-well plate containing bacterial cells, mix well, and react at 30°C for 5 min. Stop the reaction by incubating on ice for 10 min. Add 5 μL of the reaction mixture to 70 μL of 2 M sodium hydroxide, then add 25 μL of 2% salicylaldehyde, mix well, and let stand at room temperature for 1 h. Measure the absorbance at 465 nm. (Using recombinant bacteria...) E.coli The reaction of BL21(DE3) / pET28a-VmTPL was used as a control, and the absorbance ratio was taken. E.coliThe enzyme activity of mutant strains with low BL21(DE3) / pET28a-VmTPL response was accurately measured.

[0043] 4. Precise determination of positive transformant enzyme activity and phenol tolerance The procedure was the same as in Example 1, “Comparison of recombinant tyrosine phenol lyase enzyme activity and phenol tolerance”.

[0044] The results of this embodiment are as follows: Of the 482 recombinant transformant strains initially screened, 3 mutant strains with increased enzyme activity were selected. Further precise enzyme activity determination was then performed on these mutants, and the specific results are shown in Table 2. Analysis determined that the reason why the enzyme activity and phenol tolerance of the remaining 479 recombinant strains remained unchanged or decreased was due to a mutation at position 51 (aspartic acid (D) in which an amino acid other than N, T, and Q was replaced.

[0045] Table 2 Enzyme activity assay of single-point mutant recombinant bacteria

[0046] The VmTPL mutant-D51T with the highest increase in enzyme activity was designated as VmTPL-1, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-VmTPL-1.

[0047] Example 3 This example demonstrates the construction and screening of VmTPL two-site mutants. Based on the single mutant VmTPL-1 sequence constructed in Example 2, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-VmTPL-1 as a template, a single mutation was introduced at position 70. The primers are as follows: Forward primer 70E: TGGCGACNNNGCATACGCGGGTAGCAAAGACT- SEQ ID NO.11; Reverse primer 70E: CGTATGCNNNGTCGCCAGTCATCAGTGCCGCC -SEQ ID NO.12.

[0048] PCR reaction system: 25 μL of 2×FastPfu Fly Reaction Mix, 2 μL of forward primer 70E (10 μM), 2 μL of reverse primer 70E (10 μM), 1 μL of template DNA, 1 μL of FastPfu Fly DNA Polymerase, and ddH2O added to 50 μL.

[0049] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 2 min) 30 cycles; 72℃ for 10 min.

[0050] The PCR results were verified by agarose gel electrophoresis. The PCR product was then digested with DpnI enzyme at 37°C for 1 h and inactivated at 65°C for 1 min. 10 μL of the PCR product was added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "high-throughput screening of positive transformants" in Example 2).

[0051] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the enzyme activity of recombinant tyrosine phenol lyase and phenol tolerance”).

[0052] The results of this embodiment are as follows: 412 recombinant transformant strains were initially screened, and 5 mutant strains with increased enzyme activity were selected. Further precise determination of their enzyme activity was performed, and the specific results are shown in Table 3. Analysis determined that the reason why the enzyme activity and phenol tolerance of the remaining 407 recombinant strains remained unchanged or decreased was due to the mutation of glutamic acid (E) at position 70 into an amino acid other than V, A, M, S, and D.

[0053] Table 3 Enzyme activity assay of recombinant bacteria with double-point mutations

[0054] The VmTPL mutant with the highest increase in enzyme activity, -D51T-E70M, was designated VmTPL-2, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-VmTPL-2.

[0055] Example 4 Construction and screening of VmTPL three-point mutants Based on the double mutant VmTPL-2 sequence constructed in Example 3, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-VmTPL-2 as a template, a single mutation was introduced at position 72. The primers are as follows: Forward primer 72Y: CATGGCANNNGCGGGTAGCAAAGACTTCTACC- SEQ ID NO.13; Reverse primer 72Y: TACCCGCNNNTGCCATGTCGCCAGTCATCAGT- SEQ ID NO.14.

[0056] PCR reaction system: 2×FastPfu Fly Reaction Mix 25 μL, forward primer 72Y (10 μM) 2 μL, reverse primer 72Y (10 μM) 2 μL, template DNA 1 μL, FastPfu Fly DNA Polymerase 1 μL, add ddH2O to 50 μL.

[0057] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 2 min) 30 cycles; 72℃ for 10 min.

[0058] The PCR results were verified by agarose gel electrophoresis. The PCR product was then digested with DpnI enzyme at 37°C for 1 h and inactivated at 65°C for 1 min. 10 μL of the PCR product was added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "high-throughput screening of positive transformants" in Example 2).

[0059] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the enzyme activity of recombinant tyrosine phenol lyase and phenol tolerance”).

[0060] The results of this embodiment are as follows: Of the 523 recombinant transformant strains initially screened, 3 mutant strains with increased enzyme activity were selected. Further precise enzyme activity determination was then performed on these mutants, and the specific results are shown in Table 4. Analysis determined that the reason the enzyme activity and phenol tolerance of the remaining 520 recombinant strains remained unchanged or decreased was due to a mutation at position 72 (tyrosine (Y) in which an amino acid other than H, P, and F was replaced.

[0061] Table 4 Enzyme activity assay of the three-point mutant recombinant bacteria

[0062] The VmTPL mutant with the highest increase in enzyme activity, -D51T-E70M-Y72H, was designated VmTPL-3, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-VmTPL-3.

[0063] Example 5 Construction and screening of VmTPL four-site mutants Based on the triple mutant VmTPL-3 sequence constructed in Example 4, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-VmTPL-3 as a template, a single mutation was introduced at position 260. The primers are as follows: Forward primer 260K: TTCTGCCNNNAAGGACGGTCTGCAGAATTGTG- SEQ ID NO.15; Reverse primer 260K: CGTCCTTNNNGGCAGAACAGGTTGCACCATCC- SEQ ID NO.16.

[0064] PCR reaction system: 2×FastPfu Fly Reaction Mix 25 μL, forward primer 260K (10 μM) 2 μL, reverse primer 260K (10 μM) 2 μL, template DNA 1 μL, FastPfu Fly DNA Polymerase 1 μL, add ddH2O to 50 μL.

[0065] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 2 min) 30 cycles; 72℃ for 10 min.

[0066] The PCR results were verified by agarose gel electrophoresis. The PCR product was then digested with DpnI enzyme at 37°C for 1 h and inactivated at 65°C for 1 min. 10 μL of the PCR product was added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "high-throughput screening of positive transformants" in Example 2).

[0067] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the enzyme activity of recombinant tyrosine phenol lyase and phenol tolerance”).

[0068] The results of this embodiment are as follows: 389 recombinant transformant strains were initially screened, and 3 mutant strains with increased enzyme activity were selected. Further precise determination of their enzyme activity was performed, and the specific results are shown in Table 5. Analysis determined that the reason why the enzyme activity and phenol tolerance of the remaining 386 recombinant strains remained unchanged or decreased was due to a mutation at position 260 (lysine (K) in which an amino acid other than L, V, and M was replaced.

[0069] Table 5 Enzyme activity assay of the four-point mutant recombinant bacteria

[0070] The VmTPL mutant with the highest increase in enzyme activity, -D51T-E70M-Y72H-K260V, was designated VmTPL-4, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-VmTPL-4.

[0071] Example 6 Construction and screening of VmTPL five-site mutants Based on the quadruple mutant VmTPL-4 sequence constructed in Example 5, site-directed mutagenesis primers were designed. Using rapid PCR technology, with recombinant pET28a-VmTPL-4 as a template, a single mutation was introduced at position 299. The primers are as follows: Forward primer 299T: TGGTATGNNNGGTCGTGATATGTCCGCTCTGG- SEQ ID NO.17; Reverse primer 299T: CACGACCNNNCATACCACCATAGGACGGCATA- SEQ ID NO.18.

[0072] PCR reaction system: 2×FastPfu Fly Reaction Mix 25 μL, forward primer 299T (10 μM) 2 μL, reverse primer 299T (10 μM) 2 μL, template DNA 1 μL, FastPfu Fly DNA Polymerase 1 μL, add ddH2O to 50 μL.

[0073] PCR amplification conditions: 95℃ for 5 min; (95℃ for 20 s, 60℃ for 15 s, 72℃ for 2 min) 30 cycles; 72℃ for 10 min.

[0074] The PCR results were verified by agarose gel electrophoresis. The PCR product was then digested with DpnI enzyme at 37°C for 1 h and inactivated at 65°C for 1 min. 10 μL of the PCR product was added to... E.coli In BL21(DE3) competent cells, heat shock transformation was performed, followed by incubation at 37°C and 200 rpm for 1 h. The bacterial culture was then plated and incubated at 37°C for 12 h. The mutants were then subjected to initial screening (the procedure was the same as "high-throughput screening of positive transformants" in Example 2).

[0075] The wet bacterial cells were ultrasonically disrupted, and the enzyme activity was accurately measured (the procedure was the same as in Example 1, “Comparison of the enzyme activity of recombinant tyrosine phenol lyase and phenol tolerance”).

[0076] The results of this embodiment are as follows: Of the 455 recombinant transformant strains initially screened, 3 mutant strains with increased enzyme activity were identified. Further precise determination of their enzyme activity was then performed, and the specific results are shown in Table 6. Analysis determined that the reason why the enzyme activity and phenol tolerance of the remaining 452 recombinant strains remained unchanged or decreased was due to a mutation at position 299 (threonine (T) in which an amino acid other than K, F, and Y was replaced.

[0077] Table 6 Enzyme activity assay of the five-point mutant recombinant bacteria

[0078] The VmTPL mutant with the highest increase in enzyme activity, -D51T-E70M-Y72H-K260V-T299K, was designated VmTPL-5, and recombinant bacteria were obtained. E.coli BL21(DE3) / pET28a-VmTPL-5.

[0079] Example 7 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL. According to the induction expression method described in Example 1, E.coli After BL21(DE3) / pET28a-VmTPL induction and expression, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, the cells were ultrasonically disrupted to obtain VmTPL supernatant. In a 50 mL system, the following were added: VmTPL wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and conversion time 2 h. HPLC analysis showed that the remaining phenol was 69.1 g / L, the L-tyrosine yield was 19.8 g / L, and the conversion rate was 12.9%.

[0080] Example 8 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL-1. According to the induction expression method described in Example 1, E.coli After BL21(DE3) / pET28a-VmTPL-1 expression was induced, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, both bacterial cells were ultrasonically disrupted to obtain VmTPL-1 supernatant. In a 50 mL system, the following were added: VmTPL-1 wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, conversion time 2 h. HPLC analysis showed that the remaining phenol was 57.9 g / L, the L-tyrosine yield was 42.1 g / L, and the conversion rate was 27.3%.

[0081] Example 9 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL-2. According to the induction expression method described in Example 1, E.coliAfter BL21(DE3) / pET28a-VmTPL-2 expression was induced, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, both bacterial cells were ultrasonically disrupted to obtain VmTPL-2 supernatant. In a 50 mL system, the following were added: VmTPL-2 wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, conversion time 2 h. HPLC analysis showed that the remaining phenol was 48.8 g / L, the L-tyrosine yield was 59.5 g / L, and the conversion rate was 38.6%.

[0082] Example 10 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL-3. According to the induction expression method described in Example 1, E.coli After BL21(DE3) / pET28a-VmTPL-3 expression was induced, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, both bacterial cells were ultrasonically disrupted to obtain VmTPL-3 supernatant. In a 50 mL system, the following were added: VmTPL-3 wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, conversion time 2 h. HPLC analysis showed that the remaining phenol was 32.9 g / L, the L-tyrosine yield was 90.1 g / L, and the conversion rate was 58.4%.

[0083] Example 11 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL-4. According to the induction expression method described in Example 1, E.coli After BL21(DE3) / pET28a-VmTPL-4 expression was induced, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, both bacterial cells were ultrasonically disrupted to obtain VmTPL-4 supernatant. In a 50 mL system, the following were added: VmTPL-4 wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, conversion time 2 h. HPLC analysis showed that the remaining phenol was 17.1 g / L, the L-tyrosine yield was 120.8 g / L, and the conversion rate was 78.4%.

[0084] Example 12 L-tyrosine was prepared by converting pyruvate, ammonia, and phenol using the tyrosine phenol lyase VmTPL-5. According to the induction expression method described in Example 1, E.coli After BL21(DE3) / pET28a-VmTPL-5 expression was induced, bacterial cells were collected. Following the ultrasonic disruption method described in Example 1, both bacterial cells were ultrasonically disrupted to obtain VmTPL-5 supernatant. In a 50 mL system, the following were added: VmTPL-5 wet weight (added as supernatant) 5 g / L, phenol 80 g / L, sodium pyruvate 95 g / L, ammonium acetate 70 g / L, pyridoxal phosphate 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, conversion time 2 h. HPLC analysis showed that the remaining phenol was 0.8 g / L, the L-tyrosine yield was 152.3 g / L, and the conversion rate was 98.9%.

[0085] 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, The tyrosine lyase mutant has an amino acid mutation in the amino acid sequence shown in SEQ ID NO. 3, wherein the mutation site is at least one of the following positions: position 51, position 70, position 72, position 260, and position 299.

2. The tyrosine phenol lyase mutant according to claim 1, characterized in that, The tyrosine phenol lyase mutant is any one or a combination of the following (1)-(5): (1) Mutate the aspartic acid at position 51 of the amino acid sequence shown in SEQ ID NO.3 to any one of asparagine, threonine or glutamine; (2) Mutate the glutamic acid at position 70 of the amino acid sequence shown in SEQ ID NO.3 to any one of valine, alanine, methionine, serine or aspartic acid; (3) Mutate the tyrosine at position 72 of the amino acid sequence shown in SEQ ID NO.3 to any one of histidine, proline or phenylalanine; (4) Mutate the lysine at position 260 of the amino acid sequence shown in SEQ ID NO.3 to any one of leucine, valine or methionine; (5) Mutate the threonine at position 299 in the amino acid sequence shown in SEQ ID NO.3 to any one of lysine, phenylalanine or tyrosine.

3. The tyrosine phenol lyase mutant according to claim 2, characterized in that, The mutation mode of the tyrosine phenol lyase mutant is any one or a combination of the following (1)-(5): (1) Mutate the aspartic acid at position 51 of the amino acid sequence shown in SEQ ID NO.3 to threonine; (2) Mutate the glutamic acid at position 70 of the amino acid sequence shown in SEQ ID NO.3 to methionine; (3) Mutate the tyrosine at position 72 of the amino acid sequence shown in SEQ ID NO.3 to histidine; (4) Mutate the lysine at position 260 of the amino acid sequence shown in SEQ ID NO.3 to valine; (5) The threonine at position 299 in the amino acid sequence shown in SEQ ID NO.3 is mutated to tyrosine.

4. The aldose reductase mutant according to claim 3, characterized in that, The amino acid sequence of the tyrosine phenol lyase mutant is shown in SEQ ID NO.

7.

5. A biomaterial relating to the tyrosine lyase mutant according to any one of claims 1-4, characterized in that, It can be any one of the following (1)-(4): (1) A nucleic acid molecule encoding the tyrosine phenol lyase mutant according to any one of claims 1-4; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) Recombinant bacteria containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

6. A whole-cell catalyst, characterized in that, The whole-cell catalyst includes the recombinant bacteria described in claim 5.

7. The use of the tyrosine phenol lyase mutant as described in any one of claims 1-4 or the biomaterial as described in claim 5 in the synthesis of L-tyrosine.

8. A method for synthesizing L-tyrosine, characterized in that, include: The recombinant bacteria expressing the tyrosine phenol lyase mutant according to any one of claims 1-4 were cleaved, and then the supernatant was added to the transformation system to react and obtain L-tyrosine.

9. The method according to claim 8, characterized in that, The host of the recombinant bacteria includes Escherichia coli.

10. The method according to claim 8, characterized in that, The transformation system includes: phenol, sodium pyruvate, ammonium acetate, pyridoxal phosphate, phosphate buffer, and recombinant bacterial cells; Preferably, the transformation system comprises: 2-20 g / L phenol, 3-30 g / L sodium pyruvate, 2-20 g / L ammonium acetate, 0.02-0.2 g / L pyridoxal phosphate, 20-150 mM phosphate buffer, and 2-10 g / L recombinant bacterial cells; Preferably, the reaction conditions are: pH 7.5-8.5, temperature 25-35℃, shaking speed 150-250 rpm, and reaction time 1-5 h.

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