Ammonia lyase and use thereof

Abstract

The present invention provides a novel ammonia lyase. More specifically, the present invention provides a protein comprising an amino acid sequence having 80% or more sequence homology to the amino acid sequence represented by SEQ ID NO: 1 and having ammonia lyase activity.

Description

Ammonia lyase and its uses REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority based on Japanese Patent Application No. 2023-151690 filed on September 19, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an ammonia lyase and its use.

[0003] Ammonia lyases derived from various organisms are known.

[0004] According to Non-Patent Document 1, aromatic ammonia-lyases in plants include enzymes that act only on phenylalanine (Phe) or on both Phe and tyrosine (Tyr), but there have been no reports of histidine (His) ammonia-lyases (although there have been reports in animals and microorganisms). Furthermore, no ammonia-lyases that act only on Tyr have been found in plants. Furthermore, Non-Patent Document 1 discloses that the specificity of each enzyme is determined by the amino acid residues 148, 276, 405, 415, and 453. The authors also disclose that their three-dimensional structures have been analyzed, and the amino acids involved in each substrate have been identified.

[0005] Non-Patent Document 2 analyzes the three-dimensional structure of plant-derived Phe ammonia-lyase and discloses that, based on the alignment of related amino acids, activity is affected by the amino acids at positions 89 and 90. The document also discloses that His, Phe, and Cys (cysteine) are present at position 89, and Leu (leucine), Glu (glutamic acid), and His are present at position 90, and that many other conserved sequences are also present.

[0006] Non-Patent Document 3 is a document about the first TAL enzyme discovered in a microorganism. According to this document, Rhodobacter capsulatus is known to produce a substance called photoactive yellow protein, and it was speculated that TAL enzymes are involved in its biosynthesis. The document then discovered a gene sequence that shared 32% homology with PAL deduced from the genome sequence, and disclosed that this sequence was introduced into Escherichia coli, where it was expressed and the enzyme activity was compared. As a result, activity against Tyr was confirmed, and slight activity against Phe was also observed.

[0007] Non-Patent Document 4 discloses that 46 sequences presumed to be TAL enzyme genes were selected from genes registered in BLAST, and were expressed in Escherichia coli, and the activity of five of the TAL enzymes was successfully measured. The document discloses that the TAL enzyme derived from Chryseobacterium luteum sp. nov was particularly highly active, producing 2.03 g / L of coumaric acid in an 8-hour reaction, and that the TAL enzyme derived from Rivularia sp. PCC7116 produced 2.35 g / L of coumaric acid in a 24-hour reaction and also had a slight effect on Phe.

[0008] Jaime Barros and Richard A. Dixon: Plant Phenylalanie / Tyrosine Ammonia-lyases. Trends in Plant Science, (2020) 25, 66-79Gordon V Louie, Marianne E Bowman, Machelle C Mffitt, Thomas J Baiga, Bradley S Moore, and Joseph P Noel: Structural Determinants and Modulation of Substrate Specificity in Phenylalanine-Tyrosine Ammonia-Lyases. Chemistry & Biology, (2006) 13, 1327-1338JA Kyndt, TE Meyer, MA Cusanovich, JJ Van Beeumen: Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Letters, (2002) 512, 240-244Yannik Brack, Chenghai Sun, Dong Yi, Uwe T Bornscheuer: Discovery of Novel Tyrosine Ammonia Lyases for the Enzymatic Synthesis of p-Coumaric Acid. ChemBioChem. (2022) 23, e202200062

[0009] However, there remains a need for other ammonia lyases that are different from the conventional ones.

[0010] An object of the present invention is to provide an ammonia lyase and uses thereof.

[0011] As a result of extensive investigations, the present inventors have found that a protein derived from Pseudomonas chlororaphis has ammonia-lyase activity. The present invention is based on this finding.

[0012] One aspect of the present invention provides a protein having ammonia-lyase activity, comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The ammonia-lyase activity is preferably at least one activity selected from the group consisting of 4-aminophenylalanine-ammonia-lyase activity, tyrosine-ammonia-lyase activity, histidine-ammonia-lyase activity, and phenylalanine-ammonia-lyase activity.

[0013] According to other aspects of the present invention, there are provided nucleic acids encoding the above proteins, vectors comprising the nucleic acids, and cells comprising the nucleic acids or vectors in an expressible manner.

[0014] According to another aspect of the present invention, there is provided a method for producing 4-aminocinnamic acid (4-ACA), which method comprises the step of performing an enzymatic reaction using the protein of the present invention with 4-aminophenylalanine as a substrate.

[0015] According to another aspect of the present invention, there is provided a method for producing p-coumaric acid, which method comprises the step of carrying out an enzymatic reaction using tyrosine as a substrate by the protein of the present invention.

[0016] According to another aspect of the present invention, there is provided a method for producing 4-imidazole-acrylic acid, which method comprises the step of carrying out an enzymatic reaction using histidine as a substrate by the protein of the present invention.

[0017] According to another aspect of the present invention, there is provided a method for producing cinnamic acid, which method comprises the step of carrying out an enzymatic reaction using phenylalanine as a substrate by the protein of the present invention.

[0018] According to the present invention, there are provided an ammonia lyase and uses thereof.

[0019] FIG. 1 is a chromatogram showing the production of 4-aminocinnamic acid (4-ACA) when an enzymatic reaction is carried out using 4-aminophenylalanine as a substrate. From top to bottom, the reaction mixture before the reaction, a mixture containing E. coli cells and 4-aminocinnamic acid, the reaction mixture after 8 hours of reaction, and the reaction mixture after 3 hours of reaction are shown. In the reaction mixture before the reaction, a peak corresponding to the substrate 4-aminophenylalanine was observed. The peak corresponding to 4-aminocinnamic acid was larger after 8 hours of reaction than after 3 hours of reaction. FIG. 2 is a chromatogram showing the production of 4-imidazole-acrylic acid when an enzymatic reaction is carried out using histidine as a substrate. Specific Description of the Invention

[0020] According to the present invention, a protein having ammonia lyase activity is provided. This ammonia lyase enables efficient production of 4-aminocinnamic acid (4-ACA). This ammonia lyase can also be used to efficiently produce p-coumaric acid. Furthermore, this ammonia lyase can also be used to efficiently produce 4-imidazole-acrylic acid. Furthermore, this ammonia lyase can also be used to efficiently produce cinnamic acid.

[0021] The protein of the present invention comprises an amino acid sequence having 80% or more, preferably 90% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and has ammonia-lyase activity. The ammonia-lyase activity is preferably at least one activity selected from the group consisting of 4-aminophenylalanine-ammonia-lyase activity, tyrosine-ammonia-lyase activity, histidine-ammonia-lyase activity, and phenylalanine-ammonia-lyase activity.

[0022] SEQ ID NO: 1 is the amino acid sequence of a protein (PcHAL3) derived from Pseudomonas chlororaphis. The nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 2. The present invention encompasses not only PcHAL3 having the amino acid sequence shown in SEQ ID NO: 1, but also proteins that contain an amino acid sequence that has 80% or more, preferably 90% or more, sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and that have at least one of the activities exhibited by PcHAL3.

[0023] Of the above activities, 4-aminophenylalanine-ammonia-lyase activity is the activity of producing 4-aminocinnamic acid (4-ACA) using 4-aminophenylalanine as a substrate. Furthermore, tyrosine-ammonia-lyase activity is the activity of producing p-coumaric acid using tyrosine as a substrate. Furthermore, histidine-ammonia-lyase activity is the activity of producing 4-imidazole-acrylic acid using histidine as a substrate. Furthermore, phenylalanine-ammonia-lyase activity is the activity of producing cinnamic acid using phenylalanine as a substrate.

[0024] According to one embodiment of the present invention, the protein of the present invention has ammonia lyase activity. According to another embodiment of the present invention, the protein of the present invention has 4-aminophenylalanine-ammonia lyase activity. According to another embodiment of the present invention, the protein of the present invention has tyrosine-ammonia lyase activity. According to another embodiment of the present invention, the protein of the present invention has histidine-ammonia lyase activity. According to another embodiment of the present invention, the protein of the present invention has phenylalanine-ammonia lyase activity.

[0025] According to a preferred embodiment of the present invention, the protein of the present invention has 4-aminophenylalanine-ammonia-lyase activity and tyrosine-ammonia-lyase activity, and more preferably has 4-aminophenylalanine-ammonia-lyase activity, tyrosine-ammonia-lyase activity, histidine-ammonia-lyase activity and phenylalanine-ammonia-lyase activity.

[0026] Comparison of the amino acid sequence of PcHAL3 (SEQ ID NO: 1) with the amino acid sequences of ammonia lyases derived from other organisms reveals that the presence of a phenylalanine (F) residue at residue 90 and a histidine (H) residue at residue 91 in SEQ ID NO: 1 is a characteristic not found in other ammonia lyases. Based on the three-dimensional structure of the protein, residues 90 and 91 are believed to be substrate recognition sites, and it is believed that the unique substrate specificity of PcHAL3 may be attributable to these residues. Therefore, according to a preferred embodiment of the present invention, the residue corresponding to residue 90 in SEQ ID NO: 1 in the amino acid sequence of the protein of the present invention is a phenylalanine (F) residue. According to a further preferred embodiment of the present invention, the residue corresponding to residue 90 in SEQ ID NO: 1 in the amino acid sequence of the protein of the present invention is a phenylalanine (F) residue, and the residue corresponding to residue 91 in SEQ ID NO: 1 is a histidine (H) residue.

[0027] The protein of the present invention comprises an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1. Here, the sequence identity of the amino acid sequence can be determined using an algorithm known in the art, such as BLASTP, with default settings.

[0028] According to a particularly preferred embodiment of the present invention, the protein of the present invention comprises the amino acid sequence shown in SEQ ID NO:1.

[0029] The nucleic acids of the present invention encode the proteins of the present invention (their amino acid sequences). The type of nucleic acid is not particularly limited and may be DNA, RNA, or a combination thereof, but is preferably DNA. The specific nucleotide sequence of the nucleic acids of the present invention is not particularly limited as long as they encode the proteins of the present invention (their amino acid sequences). For example, the nucleic acids of the present invention may include the nucleotide sequence represented by SEQ ID NO: 2, or may include a nucleotide sequence having 80% or more, preferably 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity to the nucleotide sequence represented by SEQ ID NO: 2. Here, the sequence identity of the nucleotide sequence can be determined using algorithms known in the art, such as BLASTN, with default settings. The nucleic acids of the present invention can also be introduced into host cells and expressed in those cells. In such cases, the nucleic acids may be designed to contain many preferred codons depending on the type of host cell.

[0030] The vector of the present invention comprises the nucleic acid of the present invention. A vector is a plasmid used to introduce an expression nucleic acid construct, such as a gene, into a host cell, and various vectors are known depending on the type of host cell used. In the present invention, an appropriate vector can be selected depending on the type of host cell used. Furthermore, the nucleic acid of the present invention can be incorporated into a vector so as to be expressible in the host cell, and this can be done appropriately by those skilled in the art. For example, when the host cell is a prokaryote such as a bacterium, the vector can be one that is capable of autonomous replication in the host cell or integration into a chromosome and contains a promoter located in a position that allows transcription of the inserted nucleic acid. For example, the vector can be used to construct a series of constructs consisting of a promoter, a ribosome binding sequence, the gene, and a transcription termination sequence within the host cell.

[0031] The cells of the present invention are cells that contain the nucleic acid of the present invention or the vector of the present invention in an expressible manner. Such cells can be produced using the vector constructed as described above, depending on the type of cell used.

[0032] The type of cell (host cell) is not particularly limited, and may be either a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include bacteria and actinomycetes. Examples of eukaryotic cells include yeast, filamentous fungi, eukaryotic microalgae, plant cells, and animal cells. For reasons of ease of handling and expression efficiency, the cells used in the present invention are preferably bacteria or yeast, more preferably bacteria, and even more preferably Escherichia coli.

[0033] The present invention provides methods for producing useful substances using the protein of the present invention. Specifically, the following methods are provided: (i) a method for producing 4-aminocinnamic acid (4-ACA), comprising a step of performing an enzymatic reaction using the protein of the present invention with 4-aminophenylalanine as a substrate; (ii) a method for producing p-coumaric acid, comprising a step of performing an enzymatic reaction using the protein of the present invention with tyrosine as a substrate; (iii) a method for producing 4-imidazole-acrylic acid, comprising a step of performing an enzymatic reaction using the protein of the present invention with histidine as a substrate; and (iv) a method for producing cinnamic acid, comprising a step of performing an enzymatic reaction using the protein of the present invention with phenylalanine as a substrate.

[0034] The above-mentioned enzymatic reaction can be carried out by methods known to those skilled in the art. For example, the reaction can be carried out by adding the substrate to the medium when culturing cells (e.g., bacteria) expressing the protein of the present invention; by adding cells expressing the protein of the present invention or a lysate of such cells to a substrate solution; by adding an isolated protein of the present invention to a substrate solution; or by contacting a substrate solution with a support (e.g., a column) on which an isolated protein of the present invention has been immobilized. The temperature and pH conditions used in the reaction can be determined with reference to the optimum temperature and pH for the enzyme of the protein used, or with reference to the temperature and pH suitable for culturing the expressing cells (e.g., bacteria). According to a preferred embodiment of the present invention, the protein of the present invention used in the enzymatic reaction is supplied by the cells of the present invention.

[0035] The following table summarizes the sequence information referred to herein.

[0036] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] When the amino acid sequence of a protein derived from Pseudomonas chlororaphis (PcHAL3; its amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence encoding it is shown in SEQ ID NO:2) was compared with the amino acid sequences of ammonia lyases derived from other organisms, it was found that the presence of a phenylalanine (F) residue at residue 90 and a histidine (H) residue at residue 91 in SEQ ID NO:1 is a characteristic not found in other ammonia lyases. Based on the three-dimensional structure of the protein, residues 90 and 91 are thought to be substrate recognition sites, suggesting that this protein (PcHAL3) may be a novel ammonia lyase with substrate specificity distinct from other ammonia lyases.

[0038] In this example, the above protein (PcHAL3) was biosynthesized by recombinant expression in host cells, and its ammonia-lyase activity was examined. Escherichia coli was used as the host cell.

[0039] (1) Enzyme activity using 4-aminophenylalanine and histidine as substrates The enzymatic activity of PcHAL3 was measured using 4-aminophenylalanine and histidine as substrates. The measurement conditions and results are shown below.

[0040] Measurement conditions Reagents: 4-Amino Phenylalanine; 4-Amino Cinnamic Acid; Histidine; 4-Imidazoleacrylic Acid. Reaction solution: 10 mM 4APhe (100 mM Tris-HCl buffer, pH 8.6) (substrate solution), 10 mM His (100 mM Tris-HCl buffer, pH 8.6) (substrate solution). E. coli was concentrated with Tris-HCl to an OD600 of 10. (Specifically, the E. coli culture had an OD600 of approximately 2-3, and the culture was centrifuged to precipitate the bacteria, and Tris-HCl was added to a final OD600 of 10.) Reaction conditions: The above-mentioned E. coli cells expressing PcHAL3 were added to a 10 mM 4APhe substrate solution or a 10 mM His substrate solution, and the reaction was carried out at 37°C for 3-8 hours. (When histidine was used as the substrate, the reaction was carried out for 3 hours. When 4-aminophenylalanine was used as the substrate, the reaction was carried out twice, with reaction times of 3 and 8 hours, respectively.) Stirring speed: 450 rpm Sample preparation: Bacteria were removed by centrifugation, and the supernatant was measured. HPLC: Column: C18 (4.6 x 150 mm / 4.6 x 250 mm). Mobile phase: A: 20 mM potassium phosphate buffer; B: methanol. Flow rate: 0.8 ml / min. Program: 2% methanol for 7 min → gradient to 50% methanol in 5 min → 98% methanol for 2 min → maintain for 4 min and then stop. Detection: 280 nm / 210 nm. Sample volume: 5 μl

[0041] The results are shown in Figures 1 and 2. These figures demonstrate that 4-aminocinnamic acid (4-ACA) was efficiently produced when 4-aminophenylalanine was used as a substrate, and 4-imidazole-acrylic acid was efficiently produced when histidine was used as a substrate.

[0042] (2) Enzyme activity using phenylalanine and tyrosine as substrates The enzymatic activity of PcHAL3 was measured using phenylalanine and tyrosine as substrates. The measurement conditions and results are shown below.

[0043] Measurement conditions: Reagents: Phenylalanine; Cinnamic Acid; Tyrosine; p-Coumaric Acid. Reaction solution: 10 mM phenylalanine (100 mM Tris-HCl buffer, pH 8.5) (substrate solution), 1 mM tyrosine (70 mM sodium pyrophosphate buffer, pH 9.3) (substrate solution). E. coli cells were concentrated in buffer to an OD600 of 10. Reaction conditions: The reaction solution was prepared by adding the above E. coli cells expressing PcHAL3 to 10 mM phenylalanine or 1 mM tyrosine substrate solution, and the reaction was carried out at 37°C for 1 hour. Stirring speed: 450 rpm. Sample preparation: Bacteria were removed by centrifugation, and the supernatant was measured. HPLC: Column: C18 (4.6 x 150 mm / 4.6 x 250 mm). Mobile phase: A: 20 mM potassium phosphate buffer; B: methanol. Flow rate: 0.8 ml / min. Program: 2% methanol for 7 min → gradient to 50% methanol in 5 min → 98% methanol for 2 min → hold for 4 min and then stop. Detection: 280 nm. Sample volume: 5 μl.

[0044] result

[0045] From the above table, it can be seen that when phenylalanine is used as a substrate, cinnamic acid is efficiently produced, and when tyrosine is used as a substrate, p-coumaric acid is efficiently produced.

Claims

1. A protein comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO:1 and having ammonia lyase activity.

2. A protein comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:1 and having ammonia lyase activity.

3. The protein according to claim 1 or 2, wherein the ammonia-lyase activity is at least one activity selected from the group consisting of 4-aminophenylalanine-ammonia-lyase activity, tyrosine-ammonia-lyase activity, histidine-ammonia-lyase activity and phenylalanine-ammonia-lyase activity.

4. The protein of claim 1, wherein the residue corresponding to residue 90 in SEQ ID NO:1 is a phenylalanine (F) residue.

5. The protein of claim 1, wherein the residue corresponding to residue 90 in SEQ ID NO:1 is a phenylalanine (F) residue and the residue corresponding to residue 91 in SEQ ID NO:1 is a histidine (H) residue.

6. The protein of claim 1, which has 4-aminophenylalanine ammonia-lyase activity and tyrosine ammonia-lyase activity.

7. The protein of claim 1, which has 4-aminophenylalanine ammonia-lyase activity, tyrosine ammonia-lyase activity, histidine ammonia-lyase activity and phenylalanine ammonia-lyase activity.

8. The protein of claim 1, comprising the amino acid sequence represented by SEQ ID NO:

1.

9. A nucleic acid encoding the protein of claim 1.

10. A vector comprising the nucleic acid of claim 7.

11. A cell comprising the nucleic acid according to claim 9 or the vector according to claim 10 in an expressible manner.

12. A method for producing 4-aminocinnamic acid (4-ACA), comprising the step of carrying out an enzymatic reaction with the protein according to claim 1 using 4-aminophenylalanine as a substrate.

13. A method for producing p-coumaric acid, comprising the step of carrying out an enzymatic reaction with the protein of claim 1 using tyrosine as a substrate.

14. A method for producing 4-imidazole-acrylic acid, comprising the step of carrying out an enzymatic reaction with the protein according to claim 1 using histidine as a substrate.

15. A method for producing cinnamic acid, comprising a step of carrying out an enzymatic reaction with the protein of claim 1 using phenylalanine as a substrate.

16. The method of any one of claims 12 to 15, wherein the protein is supplied by a cell according to claim 11.

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