Modified tyrosine phenol lyase and method for producing phenol using the same

Abstract

To provide a modified tyrosine phenol lyase having improved activity for catalyzing a reaction (tyrosine cleavage reaction) for producing phenol from tyrosine.SOLUTION: Provided is a modified tyrosine phenol-lyase comprising an amino acid sequence in which one or more amino acid substitutions are introduced into an amino acid sequence of a wild-type tyrosine phenol-lyase, wherein an activity of catalyzing phenol production from tyrosine is improved compared to the wild-type tyrosine phenol-lyase, wherein the modified tyrosine phenol-lyase has amino acid substitutions at any one or more positions selected from positions corresponding to positions 25, 75, 144, 391, 419, and 456 of the amino acid sequence of SEQ ID NO: SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a modified tyrosine phenol lyase and a method for producing phenol using the same, and more particularly to a modified tyrosine phenol lyase having improved activity for catalyzing a reaction for producing phenol from tyrosine. [Background technology]

[0002] In the field of chemical production, efforts are being made to switch from petroleum-derived raw materials to biomass-derived raw materials, or from chemical synthesis methods to production methods using bioprocesses. Patent Document 1 discloses a biophenol fermentation method in which glucose is converted to tyrosine. When industrially producing phenol by fermentation, further improvement of the enzymes that catalyze each reaction from glucose to tyrosine to phenol is desired.

[0003] Tyrosine phenol lyase is an enzyme that catalyzes the reaction of synthesizing tyrosine from phenol, pyruvate, and ammonia, as well as the reverse reaction. Tyrosine phenol lyase is an industrially important enzyme used, particularly in the production of L-tyrosine and L-DOPA (see Non-Patent Document 1), and various efforts to improve its tyrosine-producing activity have been reported (see Patent Document 2). However, there are few reports on improving the phenol-producing activity (tyrosine cleavage activity) of tyrosine phenol lyase. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2023-145381 [Patent Document 2] JP 2006-320238 A [Non-patent literature]

[0005] [Non-Patent Document 1] "Simultaneous Improvement of Catalytic Activity and Thermal Stability of Tyrosine Phenol-Lyase by Directed Evolution", Eugene Rha, et. al., 2009, Vol.276, Issue 21, page 6187-6194 Summary of the Invention [Problem to be solved by the invention]

[0006] A primary object of the present disclosure is to provide a modified tyrosine phenol lyase having improved activity to catalyze a reaction that produces phenol from tyrosine (tyrosine cleavage reaction). [Means for solving the problem]

[0007] To solve the above problems, the present disclosure provides the following [1]-

[13] . [1] A modified tyrosine phenol lyase comprising an amino acid sequence in which one or more amino acid substitutions have been introduced into the amino acid sequence of a wild-type tyrosine phenol lyase, and having an improved activity for catalyzing phenol production from tyrosine compared to the wild-type tyrosine phenol lyase, A modified tyrosine phenol lyase having an amino acid substitution at one or more positions selected from the positions corresponding to the 25th, 75th, 144th, 391st, 419th and 456th positions of the amino acid sequence of SEQ ID NO: 1 when aligned with the amino acid sequence of SEQ ID NO: 1. [2] The modified tyrosine phenol lyase according to [1], which has one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of leucine at position 25 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 75 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (5) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (6) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue (excluding aspartic acid). [3] The modified tyrosine phenol lyase according to [2], which has one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of leucine at position 25 of the amino acid sequence of SEQ ID NO: 1 with lysine. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 75 of the amino acid sequence of SEQ ID NO: 1 with histidine, tyrosine, glutamine, or aspartic acid. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 is replaced with serine or leucine. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with alanine. (5) In an alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with leucine, isoleucine, or tryptophan. (6) In an alignment with the amino acid sequence of SEQ ID NO: 1, substitution of isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with threonine, lysine, or phenylalanine. [4] comprising the amino acid sequence of SEQ ID NO: 2-15; or The modified tyrosine phenol lyase according to [3], which comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequences of SEQ ID NOs: 2-15. [5] comprising the amino acid sequence of SEQ ID NO: 6, 8, 9, 10 or 13; or The modified tyrosine phenol lyase according to [3], comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 6, 8, 9, 10 or 13. [6] comprising the amino acid sequence of SEQ ID NO: 2 or 10; or The modified tyrosine phenol lyase according to [3], comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2 or 10. [7] A recombinant vector comprising a polynucleotide encoding the modified tyrosine phenol-lyase according to any one of [1] to [6]. [8] A transformant comprising the recombinant vector described in [7].

[0008] [9] A method for producing phenol, comprising a step of contacting a culture or a processed product of the culture obtained by culturing the modified tyrosine phenol lyase according to any one of [1] to [6] or the transformant according to [8] with tyrosine to obtain phenol.

[10] The method further comprises the step of obtaining tyrosine by fermenting biomass using a microorganism, The method of producing a microorganism according to [9], wherein the microorganism includes genetic modification that improves the ability to produce tyrosine from biomass.

[11] The method according to

[10] , wherein the microorganism is the transformant according to [8].

[12] The production method according to

[10] or

[11] , wherein the genetic modification that improves the ability to produce tyrosine from biomass is modification of one or more genes selected from the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase (aroG) gene, transketolase (tktA) gene, chorismate mutase / prephenate dehydrogenase (tyrA) gene, and phosphoenolpyruvate synthase (ppsA) gene.

[13] The production method according to any one of

[10] to

[12] , wherein the biomass contains one or more carbon sources selected from the group consisting of glucose, xylose, sucrose, starch, molasses, glycerol, ribitol, erythritol, and palm oil. [Effects of the Invention]

[0009] The present disclosure provides modified tyrosine phenol lyases that have improved activity to catalyze the reaction of producing phenol from tyrosine (tyrosine cleavage reaction). [Brief explanation of the drawings]

[0010] [Figure 1] The figure shows the phenol-producing activity of each modified enzyme. The horizontal axis shows each modified enzyme. The vertical axis shows the phenol concentration produced per cell weight of transformants expressing each modified enzyme, expressed as relative activity, with the value for transformants expressing the wild-type enzyme (WT) set at 1. [Figure 2] The graph shows the phenol-producing activity under conditions where phenol, the product of this reaction, was added to the reaction solution in advance (external phenol addition conditions). The horizontal axis shows each modified enzyme. The vertical axis shows the relative activity, where the concentration of phenol produced per cell weight of the transformants expressing each modified enzyme is set to 1 for the transformants expressing the wild-type enzyme (WT). DETAILED DESCRIPTION OF THE INVENTION

[0011] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the present disclosure.

[0012] 1. Modified tyrosine phenol-lyase The modified tyrosine phenol lyase according to the present disclosure comprises an amino acid sequence in which one or more amino acid substitutions have been introduced into the amino acid sequence of a wild-type tyrosine phenol lyase. The modified tyrosine phenol lyase according to the present disclosure has improved activity to catalyze the reaction of producing phenol from tyrosine (hereinafter referred to as "phenol-producing activity") compared to that of the wild-type tyrosine phenol lyase. This reaction also produces pyruvic acid and ammonia in addition to phenol. The reaction of producing phenol, pyruvic acid, and ammonia from tyrosine is shown below.

[0013] [ka]

[0014] In the present disclosure, the phenol-producing activity can be evaluated by contacting a transformant expressing the modified tyrosine phenol-lyase with tyrosine in a solvent for a predetermined period of time and then measuring the amount of phenol produced in the solvent (phenol production amount). The modified tyrosine phenol-lyase of the present invention can exhibit an increased amount of phenol produced per time per cell weight compared to the wild-type tyrosine phenol-lyase. The phenol-producing activity can also be evaluated by contacting a transformant expressing the modified tyrosine phenol-lyase with tyrosine in a solvent with exogenous phenol added for a predetermined period of time, and then measuring the amount of phenol produced in the solvent (amount of phenol produced). The modified tyrosine phenol-lyase of the present invention can exhibit an increased amount of phenol produced per time and cell weight compared to the wild-type tyrosine phenol-lyase, even under exogenous phenol added conditions.

[0015] The phenol-producing activity may be evaluated by contacting the modified tyrosine phenol-lyase with tyrosine in a solvent for a predetermined time and then measuring the amount of phenol produced in the solvent (phenol production amount). The modified tyrosine phenol-lyase of the present invention may exhibit an increased amount of phenol produced per hour compared to the wild-type tyrosine phenol-lyase. The phenol-producing activity can also be evaluated by contacting the modified tyrosine phenol-lyase with tyrosine in a solvent for a predetermined time under the condition of adding external phenol, and then measuring the amount of phenol produced in the solvent. The modified tyrosine phenol-lyase of the present invention can exhibit an increased amount of phenol produced per hour compared to the wild-type tyrosine phenol-lyase, even under the condition of adding external phenol.

[0016] In assessing phenol production activity, the reaction conditions for the substrate (tyrosine) with the modified tyrosine phenol lyase or a transformant expressing the same are, for example, in the range of a substrate concentration of 0.1-5 mM, a reaction temperature of 10-37°C, and a reaction time of 10-120 minutes. The enzymatic reaction is stopped by adding 0.1 N NaOH or 0.1 N HCl solution, after which the amount of phenol produced is quantified by LC or colorimetric assay using 4-aminoantipyrine.

[0017] The wild-type tyrosine phenol lyase into which the amino acid substitutions are introduced is not particularly limited as long as it is an enzyme having phenol-producing activity, and may be, for example, a tyrosine phenol lyase registered in a public database or a tyrosine phenol lyase described in a literature. For example, the GENBANK database provided by the National Center for Biotechnology Information (NCBI) can be used as a public database. The wild-type tyrosine phenol lyase may be an enzyme having an amino acid sequence found in an environmental metagenome and having phenol-producing activity.

[0018] Specific examples of wild-type tyrosine phenol lyase include enzymes derived from microorganisms belonging to any of the families Enterobacteriaceae, Clostridiaceae, Fusobacteriaceae, Morganellaceae, Pasteurellaceae, and Symbiobacteriaceae.

[0019] The microorganism belonging to the Enterobacteriaceae family may be a microorganism belonging to any of the genera Citrobacter, Pantoea, and Klebsiella. Microorganisms belonging to the genus Citrobacter include C. freundii, C. koseri, and C. youngae. Microorganisms belonging to the genus Pantoea include P. ananatis, P. allii, and P. agglomerans. Microorganisms belonging to the genus Klebsiella include K. oxytoca, K. africana, K. quasivariicola, and K. werkmanii.

[0020] The microorganism belonging to Clostridiaceae may be a microorganism belonging to either the genus Clostridium or the genus Tindallia. Microorganisms belonging to the genus Clostridium include C. saccharolyticum, C. tetani, C. tetanomorphum, C. malenominatum, C. cochlearium, C. liquoris, C. pascui, and C. cadaveris. Microorganisms belonging to the genus Tindallia include T. californiensis, T. magadiensis, and T. texcoconensis.

[0021] The microorganism belonging to Fusobacteriaceae may be a microorganism belonging to the genus Fusobacterium. Microorganisms belonging to Fusobacterium include F. varium, F. necrophorum, F. nucleatum, and F. russii.

[0022] The microorganism belonging to the Morganellaceae family may be a microorganism belonging to the genus Morganella. Microorganisms belonging to the genus Morganella include M. morganii and M. psychrotolerans.

[0023] The microorganism belonging to the Pasteurellaceae family may be a microorganism belonging to either the genus Aggregatibacter or the genus Pasteurella. Microorganisms belonging to the genus Aggregatibacter include A. actinomycetemcomitans, A. aphrophilus, and A. kilianii. Examples of microorganisms belonging to the genus Pasteurella include P. multocida and P. skyensis.

[0024] The microorganism belonging to the Symbiobacteriaceae may be a microorganism belonging to the genus Symbiobacterium. Microorganisms belonging to the genus Symbiobacterium include S. thermophilum, S. toebii, and S. sp.

[0025] The wild-type tyrosine phenol-lyase preferably used is the wild-type tyrosine phenol-lyase derived from Citrobacter freundii (SEQ ID NO: 1).Other wild-type tyrosine phenol-lyases preferably used include the wild-type tyrosine phenol-lyase derived from Pantoea agglomerans (SEQ ID NO: 31), the wild-type tyrosine phenol-lyase derived from Morganella morganii (SEQ ID NO: 32), and the wild-type tyrosine phenol-lyase derived from Symbiobacterium sp. (SEQ ID NO: 33).

[0026] The position at which the amino acid substitution is introduced into the wild-type tyrosine phenol-lyase is not particularly limited as long as it results in an improvement in phenol-producing activity. However, when aligned with the amino acid sequence of SEQ ID NO: 1, the substitution is preferably at one or more positions selected from the group consisting of positions 25, 75, 144, 391, 419, and 456 of the amino acid sequence of SEQ ID NO: 1. Substitution of amino acid residues at these positions in the wild-type tyrosine phenol-lyase derived from Citrobacter freundii, which comprises the amino acid sequence of SEQ ID NO: 1, altered the phenol-forming activity, revealing that the amino acid residues at these positions play an important role in determining the phenol-forming activity of tyrosine phenol-lyase. Therefore, substitution of amino acid residues at these positions with other amino acid residues can alter the phenol-forming activity of tyrosine phenol-lyase, and it is expected that modified tyrosine phenol-lyases exhibiting the desired enzymatic activity can be obtained.

[0027] Here, "the position corresponding to the 25th amino acid in the amino acid sequence of SEQ ID NO: 1 when aligned with the amino acid sequence of SEQ ID NO: 1" means the position corresponding to the 25th amino acid in the amino acid sequence of SEQ ID NO: 1 when the amino acid sequence of the wild-type tyrosine phenol-lyase into which the amino acid substitution is to be introduced is aligned with the amino acid sequence of the wild-type tyrosine phenol-lyase derived from Citrobacter freundii of SEQ ID NO: 1. The same applies to the positions corresponding to the 75th, 144th, 391st, 419th, and 456th amino acids in the amino acid sequence of SEQ ID NO: 1.

[0028] Alignment is performed by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match. During alignment, gaps are inserted as needed into one or both of the two amino acid sequences to be compared. Such amino acid sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTALW.

[0029] Specifically, the amino acid substitution in the wild-type tyrosine phenol lyase is preferably one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of leucine at position 25 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 75 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (5) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (6) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue (excluding aspartic acid).

[0030] More specifically, the amino acid substitution in the wild-type tyrosine phenol lyase is preferably one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of leucine at position 25 of the amino acid sequence of SEQ ID NO: 1 with lysine. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 75 of the amino acid sequence of SEQ ID NO: 1 with histidine, tyrosine, glutamine, or aspartic acid. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 is replaced with serine or leucine. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with alanine. (5) In an alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with leucine, isoleucine, or tryptophan. (6) In an alignment with the amino acid sequence of SEQ ID NO: 1, substitution of isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with threonine, lysine, or phenylalanine.

[0031] SEQ ID NO: 2 shows the amino acid sequence of a modified tyrosine phenol-lyase obtained by introducing the amino acid substitution V419L into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO:3 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution V419I has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO:1. SEQ ID NO: 4 shows the amino acid sequence of a modified tyrosine phenol-lyase obtained by introducing the amino acid substitution V419W into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 5 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution I456F was introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 6 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution I456T was introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 7 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution I456D has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 8 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution I456K has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 9 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution V391A was introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii of SEQ ID NO: 1. SEQ ID NO: 10 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution L25K has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 11 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution E75Q has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 12 shows the amino acid sequence of a modified tyrosine phenol-lyase obtained by introducing the amino acid substitution E75H into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 13 shows the amino acid sequence of a modified tyrosine phenol-lyase in which the amino acid substitution E75D has been introduced into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii of SEQ ID NO: 1. SEQ ID NO: 14 shows the amino acid sequence of a modified tyrosine phenol-lyase obtained by introducing the amino acid substitution E144L into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1. SEQ ID NO: 15 shows the amino acid sequence of a modified tyrosine phenol-lyase obtained by introducing the amino acid substitution E144S into the wild-type tyrosine phenol-lyase derived from Citrobacter freundii shown in SEQ ID NO: 1.

[0032] The modified tyrosine phenol lyase comprising the amino acid sequence of SEQ ID NO: 6, 8, 9, 10 or 13 can exhibit phenol production activity that is 120% or more compared to the wild-type tyrosine phenol lyase. The modified tyrosine phenol lyase comprising the amino acid sequence of SEQ ID NO: 4, 5, 11 or 15 can exhibit phenol production activity that is 110% or more compared to the wild-type tyrosine phenol lyase. The modified tyrosine phenol lyase comprising the amino acid sequence of SEQ ID NO: 2 or 10 has phenol production activity that is more than 100% of that of the wild-type tyrosine phenol lyase, and can exhibit phenol production activity that is more than 100% of that of the wild-type tyrosine phenol lyase under conditions where external phenol is added. In light of the above, it is preferable that the amino acid substitution in the wild-type tyrosine phenol lyase is, more specifically, an amino acid substitution shown in any of SEQ ID NOs: 2, 4, 5, 6, 8, 9, 10, 11, 13, or 15, from the viewpoint of phenol production activity and / or phenol production activity under conditions of external phenol addition, and of these, SEQ ID NOs: 2, 6, 8, 9, 10, or 13 is more preferable.

[0033] In one embodiment, the modified tyrosine phenol lyase according to the present disclosure comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2-15 and has phenol-producing activity. The sequence identity of the modified tyrosine phenol lyase according to the present disclosure to the amino acid sequence of SEQ ID NO: 2-15 is preferably identity to the amino acid sequence of SEQ ID NO: 1 excluding positions 25, 75, 144, 391, 419, and 456. Modified tyrosine phenol lyases comprising such amino acid sequences are expected to have improved phenol production activity and / or phenol production activity under conditions of external phenol addition, similar to modified tyrosine phenol lyases comprising the amino acid sequences of SEQ ID NOs: 2-15.

[0034] Here, the "identity" of sequences refers to, in the case of amino acid sequences, aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and dividing the number of matched amino acid residues by the total number of amino acid residues, expressed as a percentage. During the alignment, gaps may be inserted as needed into one or both of the two sequences to be compared. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTALW. When gaps are inserted, the total number of amino acid residues is calculated by counting each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the number of matched amino acid residues by the total number of amino acid residues in the longer sequence. The same applies to the identity of nucleic acid sequences.

[0035] The sequence identity of the modified tyrosine phenol lyase of the present disclosure to the amino acid sequence of SEQ ID NO: 2-15 is preferably 91% or more, 92% or more, 93% or more, more preferably 94% or more, 95% or more, 96% or more, even more preferably 97% or more, 98% or more, 99% or more, and particularly preferably 99.5% or more. The modified tyrosine phenol lyase according to the present disclosure may have a modified amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2-15, and may have phenol-producing activity. Here, several means 20 or less, preferably 10 or less, more preferably 5 or less, even more preferably 3 or less, and most preferably 2.

[0036] 2. Vectors and Transformants The present disclosure also provides a recombinant vector containing a polynucleotide encoding the above-described modified tyrosine phenol-lyase, as well as a transformant containing the recombinant vector. The term "polynucleotide encoding a modified tyrosine phenol-lyase" also includes a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a base sequence complementary to the polynucleotide encoding the modified tyrosine phenol-lyase and encodes a protein that exhibits improved phenol-producing activity compared to wild-type tyrosine phenol-lyase. The polynucleotide is particularly DNA. "Stringent conditions" refer to post-hybridization washing conditions, including a salt concentration of 300-2000 mM and a temperature of 40-75°C, preferably a salt concentration of 600-900 mM and a temperature of 65°C. Examples include 2xSSC at 50°C. Those skilled in the art can appropriately determine "stringent conditions" by taking into account not only the salt concentration of the buffer and temperature, but also other conditions such as probe concentration, probe length, and reaction time. For detailed procedures for hybridization, see "Molecular Cloning, A Laboratory Manual 2nd Ed. (Cold Spring Harbor Laboratory Press (1989)) and the like.

[0037] The polynucleotide encoding the modified tyrosine phenol-lyase can be incorporated into a recombinant vector and the recombinant vector can be introduced into a host organism by conventionally known techniques. Examples of vectors that can be used include plasmid DNA, bacteriophage DNA, retrotransposon DNA, and artificial chromosome DNA. In addition to the tyrosine phenol lyase gene, a promoter, a terminator, an enhancer, a splicing signal, a poly(A) addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like can be ligated to the vector. Examples of selection markers include the kanamycin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, and chloramphenicol resistance gene.

[0038] The host organism is not particularly limited as long as it is capable of expressing the desired tyrosine phenol lyase after the recombinant vector is introduced, and examples that can be used include bacteria such as Escherichia coli and actinomycetes, yeast, animal cells, insect cells, and plant cells. When bacteria are used as hosts, actinomycetes such as Escherichia coli, Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC17895, and Rhodococcus rhodochrous ATCC19140 are preferably used. Methods for introducing recombinant vectors into bacteria include, for example, calcium ion methods and electroporation. When Escherichia coli is used as a host, it is preferable to use expression vectors with high expression efficiency, such as the expression vectors pKK233-2 (Amersham Biosciences) or pTRC99A (Amersham Biosciences) that contain the trc promoter. When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, etc. are preferably used. Methods for introducing a recombinant vector into yeast include, for example, electroporation, the spheroplast method, and the lithium acetate method. When animal cells are used as hosts, COS-7 cells, VERO cells, CHO cells, L cells, GH3 cells, FL cells, etc. are preferably used. Methods for introducing recombinant vectors into animal cells include, for example, electroporation, calcium phosphate method, and lipofection. When insect cells are used as hosts, SF9 cells, SF21 cells, etc. are preferably used. Methods for introducing recombinant vectors into insect cells include, for example, the calcium phosphate method, lipofection, and electroporation. When plant cells are used as hosts, tobacco BY-2 cells and the like are preferably used. Methods for introducing recombinant vectors into plant cells include, for example, the Agrobacterium method, particle gun method, PEG method, and electroporation method. After transformation, plant cells may be cultured either as cells or as whole plants.

[0039] 3. Phenol production method The present disclosure also provides a method for producing phenol, comprising the step of contacting the above-mentioned modified tyrosine phenol lyase, or a culture obtained by culturing the above-mentioned transformant, or a processed product of the culture, with tyrosine to obtain phenol. The method for producing phenol according to the present disclosure may further include a step of obtaining tyrosine by fermenting biomass using a microorganism or the above-described transformant, and the microorganism or the above-described transformant may include genetic modification that improves the ability to produce tyrosine from biomass.

[0040] Here, "contact" means bringing the modified tyrosine phenol-lyase or transformant and tyrosine into the same reaction or culture system. Examples of "contact" include mixing a separated and purified modified tyrosine phenol-lyase with tyrosine, adding tyrosine to a culture vessel containing cells (transformants) expressing the modified tyrosine phenol-lyase gene, culturing the cells in the presence of tyrosine, and mixing an extract of the cells with tyrosine.

[0041] Examples of "treated culture products" include cells (transformants) after culture encapsulated in a gel such as acrylamide, treated with glutaraldehyde, or supported on an inorganic carrier such as alumina, silica, zeolite, or diatomaceous earth.

[0042] [Tyrosine manufacturing process] The substrate tyrosine is preferably L-tyrosine, which may be obtained from biomass by fermentation. Biomass refers to organic resources derived from plants and animals that can be recycled into energy or materials and excludes fossil resources. In the present disclosure, biomass particularly refers to resources containing a carbon source that can be subjected to microbial fermentation. Examples of carbon sources include, but are not limited to, fermentable carbohydrates or oils such as glucose, xylose, sucrose, starch, blackstrap molasses, glycerol, ribitol, erythritol, and palm oil. Biomass may also contain lipids, amino acids, organic acids, and alcohols that can be derived from or produced by metabolism of these carbon sources. These biomasses can be used singly or in combination of two or more.

[0043] The microorganisms used for fermentation may be any microorganisms capable of producing tyrosine from biomass. Most microorganisms have this ability, but examples of usable microorganisms include the following: Bacteria of the genus Escherichia such as Escherichia coli; bacteria of the genus Pseudomonas such as Pseudomonas putida; bacteria of the genus Corynebacterium such as Corynebacterium glutamicum; bacteria of the genus Bacillus such as Bacillus subtilis; yeasts of the genus Pichia such as Pichia pastoris; yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae; filamentous fungi of the genus Aspergillus such as Aspergillus oryzae, Aspergillus nidulans, and Aspergillus niger. The microorganism may be one selected from these, or two or more selected from these, and may be any strain, such as a mutant strain obtained by commonly used mutation treatments such as UV irradiation or EMS treatment, or a recombinant strain induced by genetic techniques such as cell fusion or gene recombination.

[0044] The microorganism or transformant used for fermentation may be a genetically modified microorganism obtained by genetically modifying the above-mentioned microorganism to enhance its ability to produce tyrosine. Genetic modifications to enhance tyrosine production include enhancing the activity of the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase (aroG) gene, transketolase (tktA) gene, chorismate mutase / prephenate dehydrogenase (tyrA) gene, and phosphoenolpyruvate synthase (ppsA) gene.

[0045] 7-Phospho-2-dehydro-3-deoxyarabinoheptonate aldolase (aroG) is an enzyme gene that produces 7-phospho-2-dehydro-3-deoxyarabinoheptonate and inorganic phosphate from D-erythrose-4-phosphate, phosphoenolpyruvate, and water. The activity of this enzyme is known to be inhibited by phenylalanine. The 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase used in the present disclosure preferably contains a mutation that eliminates the inhibition by phenylalanine. Known mutations can be used, including, for example, the D146N and F209S mutations in the Escherichia coli-derived enzyme (uniport id: P0AB91).

[0046] Transketolase (tktA) is an enzyme that converts fructose-6-phosphate and glyceraldehyde-3-phosphate into erythrose-4-phosphate and xylulose-5-phosphate.

[0047] Chorismate mutase / prephenate dehydrogenase (tyrA) is an enzyme that converts chorismate and NAD+ into 3-(4-hydroxyphenyl)pyruvate, NADH, and carbon dioxide. It is known that the activity of this enzyme is inhibited by tyrosine. The chorismate mutase / prephenate dehydrogenase used in the present disclosure preferably contains a mutation that eliminates the inhibition by tyrosine. Known mutations can be used, including, for example, the M53I and A354V mutations in the Escherichia coli-derived enzyme (uniport ID: P07023).

[0048] Phosphoenolpyruvate synthase (ppsA) is an enzyme that converts pyruvate, ATP, and water into phosphoenolpyruvate, AMP, and inorganic phosphate.

[0049] Enhancing enzyme activity includes increasing the expression level of a gene by introducing a gene encoding the enzyme, strongly expressing the gene by enhancing the promoter activity of the gene, or increasing the expression level by reducing or inactivating the repressor activity of the gene. Gene transfer and the like can be carried out according to conventionally known molecular biological techniques. Enhancement of enzyme activity and removal of feedback inhibition can be confirmed by activity measurement using a cell-free extract of the microorganism or purified enzyme.

[0050] Fermentation may be carried out by culturing microorganisms using conventionally known techniques, and tyrosine accumulates within the cells and in the culture supernatant as a result of the metabolism of biomass by the microorganisms. The aqueous medium (culture medium) containing the biomass used for cultivation is a solid or liquid medium containing sufficient nutrients, including at least one carbon source, in which the microorganisms can grow. The concentration of the biomass raw material in the medium is not particularly limited as long as it can produce tyrosine. The preferred concentration of the biomass raw material has a lower limit of typically 0.05 (w / v)% or more, more preferably 0.1 (w / v)% or more, and even more preferably 0.2 (w / v)% or more, and an upper limit of typically 20 (w / v)% or less, more preferably 15 (w / v)% or less, and even more preferably 10 (w / v)% or less. The use of a concentration of 0.05 (w / v)% or more is because it increases the tyrosine-producing ability of the microorganism, while the use of a concentration of 20 (w / v)% or less is because no significant increase in the effect is observed even if the concentration exceeds this limit. In addition, additional biomass raw material may be added in accordance with the decrease in the biomass raw material as the reaction proceeds. The produced tyrosine may be partially or completely purified from the culture medium by known techniques, or may remain unpurified.

[0051] [Phenol manufacturing process] The process for producing phenol from tyrosine by a reaction catalyzed by tyrosine phenol lyase may be a fermentation process using microorganisms (in vivo) or a cell-free system (in vitro) process not involving microbial fermentation, but a fermentation process using biomass is preferred.

[0052] When this step is performed by a cell-free process, it is preferable to recycle the enzyme catalyst as long as its activity is not inactivated. In order to prevent the enzyme from being inactivated and to facilitate recycling, it is preferable to use the enzyme catalyst in the form of a treated product.

[0053] When this step is a fermentation process using biomass, a transformant into which a modified tyrosine phenol lyase gene has been introduced is cultured in the presence of biomass by a conventionally known method, and the produced phenol accumulates within the bacterial cells and in the culture supernatant. The aqueous medium (culture medium) containing the biomass used for cultivation is a solid or liquid medium containing sufficient nutrients, including at least one carbon source, in which the microorganisms can grow. The concentration of the biomass raw material in the medium is not particularly limited as long as it can produce phenol. The preferred concentration of the biomass raw material has a lower limit of typically 0.05 (w / v)% or more, more preferably 0.1 (w / v)% or more, and even more preferably 0.2 (w / v)% or more, and an upper limit of typically 20 (w / v)% or less, more preferably 15 (w / v)% or less, and even more preferably 10 (w / v)% or less. The use of a concentration of 0.05 (w / v)% or more is due to an increase in the phenol-producing ability of the microorganism, while the use of a concentration of 20 (w / v)% or less is due to the fact that no significant increase in the effect is observed even if the concentration is higher than this. In addition, additional biomass raw material may be added in accordance with the decrease in the biomass raw material as the reaction proceeds. Furthermore, the host cell of the transformant into which the modified tyrosine phenol lyase of the present invention has been genetically introduced may be a genetically modified microorganism that has been genetically modified to enhance the above-mentioned tyrosine production ability. By using such a host cell, it is also possible to produce phenol by fermentation using a biomass fermentation process.

[0054] The produced phenol can be separated and purified from the culture or reaction solution by using well-known procedures such as filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, solvent extraction, distillation, and crystallization in appropriate combinations as needed. The phenol produced and the amount produced can be detected and measured using conventional methods such as high performance liquid chromatography and LC / MS. [Example]

[0055] 1. Preparation of Plasmids and Transformants Using a machine learning model that takes the amino acid sequence of an enzyme as input and outputs a predicted kcat value, we determined amino acid sequence substitution positions expected to improve the kcat value for wild-type tyrosine phenol-lyase (amino acid sequence: SEQ ID NO: 1, polynucleotide sequence: SEQ ID NO: 16) derived from Citrobacter freundii. Fourteen modified enzymes were created by introducing amino acid substitutions at these positions. The amino acid sequences of the modified enzymes are shown in SEQ ID NOs: 2-15, and the polynucleotide sequences are shown in SEQ ID NOs: 17-30. The polynucleotides of SEQ ID NOs: 16-30 were inserted into the NdeI-BglII site of the plasmid vector pACYCDuet-1 to obtain expression plasmids for each enzyme. E. coli BL21(DE3) was transformed with the expression plasmids according to standard methods to obtain transformants.

[0056] 2.Bacterial cell culture LB medium (containing 34 μg / mL chloramphenicol) was dispensed into a 96-deep-well plate at 250 μL / well, and the transformant was inoculated into each well and cultured overnight at 37°C and 1,200 rpm on a microplate shaker to prepare a preculture solution. 2xYT medium (containing 34 μg / mL chloramphenicol) was dispensed into a 96-deep-well plate at 250 μL / well, and 5 μL / well of the pre-culture solution was inoculated into each well. The plates were then cultured at 37°C, 1,200 rpm, and 2 hours on a microplate shaker. 250 μL of 2xYT medium (containing 34 μg / mL chloramphenicol and 0.6 mM IPTG) was added to each well, and the mixture was cultured overnight at 30°C and 1,200 rpm to prepare the main culture solution for evaluation. After the culture was completed, the turbidity (OD600) of the main culture solution was measured.

[0057] 3. Tyrosine cleavage reaction 100 mM HEPES (pH 8.0, containing 125 μM PLP) was dispensed into a 96-deep well plate at 400 μL / well, and the culture medium was dispensed into each well at 20 μL / well. 100 μL / well of 25 mM L-tyrosine (0.1 N NaOH solution) was dispensed and sealed, and then the plate was shaken on a microplate shaker at 30° C., 800 rpm, for 1 hour.

[0058] 4. Measurement of phenol-producing activity 100 mM HEPES (pH 8.0, 100 mM NaOH, containing 0.6% w / v 4-aminoantipyrine) was dispensed into a 96-well flat-bottom plate for phenol determination at 120 μL / well. After the tyrosine cleavage reaction, the 96-deep-well plate was centrifuged at 4°C, 5,000 rpm for 10 minutes, and the supernatant was dispensed into a phenol quantitation plate at 10 μL / well. After thorough mixing, the plate was allowed to stand at room temperature for 5 minutes. 30 μL of 0.6% w / v aqueous potassium peroxodisulfate solution was dispensed into each well and mixed thoroughly. After allowing to stand at room temperature for 10 minutes, the absorbance at 500 nm was measured using a microplate reader to calculate the concentration of the phenol produced. The concentration of phenol produced per cell weight was calculated from the concentration of phenol produced and the turbidity of the cells.

[0059] The phenol concentration per cell weight of the transformants expressing each modified enzyme is shown in Figure 1. The horizontal axis shows each modified enzyme, and the vertical axis shows the phenol concentration per cell weight as a relative activity, with the value for the transformant expressing the wild-type enzyme (WT) set at 1. The modified enzymes V391A, I456K, E75D, L25K, and I456T showed phenol-producing activity of 120% or more compared to the wild-type enzyme. The modified enzymes V419W, E144S, I456F, and E75Q exhibited phenol-producing activity that was 110% or higher than that of the wild-type enzyme. The modified enzymes V419I, E144L, E75H, and 419L exhibited phenol-producing activity exceeding 100% of that of the wild-type enzyme. On the other hand, the modified enzyme I456D did not show any significant activity compared to the wild-type enzyme.

[0060] 5. Evaluation of phenol production activity under external phenol addition conditions LB medium (containing 34 μg / mL chloramphenicol) was dispensed into 14 mL round-bottom tubes at 2 mL each, and the transformant was inoculated into each tube and cultured overnight at 37°C and 200 rpm in a bioshaker to prepare a preculture solution. 10 mL of 2xYT medium (containing 34 μg / mL chloramphenicol) was dispensed into 50 mL tubes, and 100 μL of the preculture solution was inoculated into each tube, followed by cultivation in a bioshaker at 30°C, 200 rpm, for 2 hours. A filter-sterilized aqueous IPTG solution was added to the tube to a final concentration of 0.3 mM, and the mixture was cultured overnight at 30°C and 200 rpm to prepare the main culture solution, which was then used for evaluation. After the cultivation was completed, the turbidity (OD600) of the main culture solution was measured.

[0061] 100 mM HEPES (pH 8.0, containing 125 μM PLP) was dispensed into a 96-deep-well plate at 350 μL / well, and 20 μL of the culture medium (adjusted to OD600 = 2 with 100 mM HEPES) was dispensed into each well. 100 μL / well of 25 mM L-tyrosine (0.1 N NaOH solution) and 50 μL / well of 15 mM phenol (100 mM HEPES solution) were dispensed and sealed. The plate was then shaken at 30°C, 800 rpm, and 2 hours on a microplate shaker. 50 μL of the reaction mixture was dispensed into a new 96-deep-well plate, and 50 μL / well of 1 N hydrochloric acid, 200 μL / well of acetonitrile, and 200 μL / well of Milli-Q water were added. After mixing thoroughly, the plate was sealed and centrifuged at 4°C, 5,000 rpm, and 10 minutes. The supernatant was then subjected to LC-MS analysis.

[0062] LC-MS analysis conditions Instrument: Waters UPLC-MS (SQD2) Column: ACQUITY UPLC-CSH C18 1.7μm, 2.1×150 mm Eluents: A) 0.1% formic acid / water, B) 0.1% formic acid / acetonitrile Detector: PDA (260-400 nm), MS (ESI+ / -) Flow rate: 0.2 mL / min Gradient %B: 0.5 minutes 10% 8.5 minutes 90% 9.5 minutes 90% 10.5 minutes 10% 12.5 minutes 10%

[0063] The phenol concentration per cell weight for the transformants expressing each modified enzyme is shown in Figure 2. The horizontal axis shows each modified enzyme, and the vertical axis shows the phenol concentration per cell weight as relative activity, with the value for the transformant expressing the wild-type enzyme (WT) set at 1. The modified enzymes V419L and L25K both exhibited phenol-producing activity exceeding 100% of that of the wild-type (see also "4. Measurement of phenol-producing activity"). This confirmed that their phenol-producing activity under exogenous phenol addition conditions was improved compared to the wild-type enzyme. In particular, the modified enzyme V419L exhibited remarkable phenol-producing activity exceeding 140% of that of the wild-type enzyme under exogenous phenol addition conditions. [Sequence List Free Text]

[0064] SEQ ID NO: 1: Amino acid sequence of the wild-type enzyme from Citrobacter freundii SEQ ID NO: 2: Amino acid sequence of modified enzyme V419L SEQ ID NO: 3: Amino acid sequence of modified enzyme V419I SEQ ID NO: 4: Amino acid sequence of modified enzyme V419W SEQ ID NO: 5: Amino acid sequence of modified enzyme I456F SEQ ID NO: 6: Amino acid sequence of modified enzyme I456T SEQ ID NO: 7: Amino acid sequence of modified enzyme I456D SEQ ID NO: 8: Amino acid sequence of modified enzyme I456K SEQ ID NO: 9: Amino acid sequence of modified enzyme V391A SEQ ID NO: 10: Amino acid sequence of modified enzyme L25K SEQ ID NO: 11: Amino acid sequence of modified enzyme E75Q SEQ ID NO: 12: Amino acid sequence of modified enzyme E75H SEQ ID NO: 13: Amino acid sequence of modified enzyme E75D SEQ ID NO: 14: Amino acid sequence of modified enzyme E144L SEQ ID NO: 15: Amino acid sequence of modified enzyme E144S SEQ ID NO: 16: Nucleotide sequence of the wild-type enzyme from Citrobacter freundii SEQ ID NO: 17: Nucleotide sequence of modified enzyme V419L SEQ ID NO: 18: Nucleotide sequence of modified enzyme V419I SEQ ID NO: 19: Nucleotide sequence of modified enzyme V419W SEQ ID NO: 20: Nucleotide sequence of the modified enzyme I456F SEQ ID NO: 21: Nucleotide sequence of modified enzyme I456T SEQ ID NO: 22: Nucleotide sequence of modified enzyme I456D SEQ ID NO: 23: Nucleotide sequence of modified enzyme I456K SEQ ID NO: 24: Nucleotide sequence of modified enzyme V391A SEQ ID NO: 25: Nucleotide sequence of the modified enzyme L25K SEQ ID NO: 26: Nucleotide sequence of modified enzyme E75Q SEQ ID NO: 27: Nucleotide sequence of modified enzyme E75H SEQ ID NO: 28: Nucleotide sequence of modified enzyme E75D SEQ ID NO: 29: Nucleotide sequence of modified enzyme E144L SEQ ID NO: 30: Nucleotide sequence of modified enzyme E144S SEQ ID NO: 31: Amino acid sequence of the wild-type enzyme from Pantoea agglomerans SEQ ID NO: 32: Amino acid sequence of the wild-type enzyme from Morganella morganii SEQ ID NO: 33: Amino acid sequence of the wild-type enzyme from Symbiobacterium sp.

Claims

1. A modified tyrosine phenol lyase comprising an amino acid sequence in which one or more amino acid substitutions have been introduced into the amino acid sequence of a wild-type tyrosine phenol lyase, and having an improved activity for catalyzing phenol production from tyrosine compared to the wild-type tyrosine phenol lyase, A modified tyrosine phenol lyase having an amino acid substitution at one or more positions selected from the positions corresponding to the 25th, 75th, 144th, 391st, 419th and 456th positions of the amino acid sequence of SEQ ID NO: 1 when aligned with the amino acid sequence of SEQ ID NO:

1.

2. The modified tyrosine phenol lyase according to claim 1, which has one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of the 25th leucine in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 75 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (5) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. (6) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of the isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with another amino acid residue.

3. The modified tyrosine phenol lyase according to claim 2, which has one or more amino acid substitutions selected from the following (1) to (6): (1) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of leucine at position 25 of the amino acid sequence of SEQ ID NO: 1 with lysine. (2) In alignment with the amino acid sequence of SEQ ID NO: 1, the 75th glutamic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with histidine, tyrosine, glutamine, or aspartic acid. (3) In alignment with the amino acid sequence of SEQ ID NO: 1, glutamic acid at position 144 of the amino acid sequence of SEQ ID NO: 1 is replaced with serine or leucine. (4) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 391 of the amino acid sequence of SEQ ID NO: 1 with alanine. (5) In an alignment with the amino acid sequence of SEQ ID NO: 1, substitution of valine at position 419 of the amino acid sequence of SEQ ID NO: 1 with leucine, isoleucine, or tryptophan. (6) In alignment with the amino acid sequence of SEQ ID NO: 1, substitution of isoleucine at position 456 of the amino acid sequence of SEQ ID NO: 1 with threonine, lysine, or phenylalanine.

4. comprising the amino acid sequence of SEQ ID NO: 2-15; or The modified tyrosine phenol-lyase according to claim 3, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequences of SEQ ID NOs: 2-15.

5. comprising the amino acid sequence of SEQ ID NO: 6, 8, 9, 10 or 13; or The modified tyrosine phenol-lyase of claim 3, comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 6, 8, 9, 10 or 13.

6. comprising the amino acid sequence of SEQ ID NO: 2 or 10; or The modified tyrosine phenol-lyase according to claim 3, comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2 or 10.

7. A recombinant vector comprising a polynucleotide encoding the modified tyrosine phenol-lyase of any one of claims 1 to 6.

8. A transformant comprising the recombinant vector according to claim 7.

9. A method for producing phenol, comprising a step of contacting a culture or a processed product of the culture obtained by culturing the modified tyrosine phenol lyase according to any one of claims 1 to 6, or the transformant according to claim 8, with tyrosine to obtain phenol.

10. The method further comprises the step of obtaining tyrosine by fermenting biomass using a microorganism, The method according to claim 9 , wherein the microorganism comprises a genetic modification that improves its ability to produce tyrosine from biomass.

11. The method according to claim 10, wherein the microorganism is the transformant according to claim 8.

12. The production method according to claim 10, wherein the genetic modification that improves the ability to produce tyrosine from biomass is a modification of one or more genes selected from the group consisting of the 7-phospho-2-dehydro-3-deoxyarabinoheptonate aldolase (aroG) gene, the transketolase (tktA) gene, the chorismate mutase / prephenate dehydrogenase (tyrA) gene, and the phosphoenolpyruvate synthase (ppsA) gene.

13. 13. The method of claim 12, wherein the biomass comprises one or more carbon sources selected from the group consisting of glucose, xylose, sucrose, starch, molasses, glycerol, ribitol, erythritol, and palm oil.

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