Method for producing β-Ala-DOPA

Abstract

To provide a technique to efficiently synthesize β-Ala-DOPA.SOLUTION: A method for producing β-Ala-DOPA includes the step (1) for using a microbe strain that expresses L-amino acid α-ligase activity, to synthesize β-Ala-DOPA from β-Ala and L-DOPA as substrates.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing β-Ala-DOPA. [Background technology]

[0002] In mammals, L-tyrosine (L-4-hydroxyphenylalanine, L-Tyr) is synthesized in the body and brain:

[0003] [ka] From there, tyrosine hydroxylase converts it into L-dopa (L-3,4-dihydroxyphenylalanine, L-DOPA, or levodopa):

[0004] [ka] are synthesized.

[0005] L-dopa is converted by levodopa decarboxylase into dopamine:

[0006] [ka] L-dopa is a precursor to the neurotransmitter dopamine.

[0007] The present inventors have developed a method for synthesizing L-carnosine (β-Ala-His) using a protein (YwfE) with L-amino acid α-ligase activity, with β-alanine and L-histidine as substrates in the presence of ATP, and for synthesizing carnosine using Escherichia coli expressing the protein (Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6934774 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a technique for efficiently synthesizing β-Ala-DOPA. [Means for solving the problem]

[0010] The present invention includes the following method for producing β-Ala-DOPA.

[0011] This technology synthesizes β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) in a single step using a protein with L-amino acid α-ligase activity in a bacterial reaction system or an enzymatic reaction system.

[0012] This technology involves two steps in a bacterial or enzymatic reaction system: (1) β-Ala-Tyr is synthesized from β-alanine (β-Ala) and L-tyrosine (L-Tyr) using a protein with L-amino acid α-ligase activity, and (2) β-Ala-DOPA is synthesized from β-Ala-Tyr using a protein with two-component flavin-dependent monooxygenase activity.

[0013] Section 1. A method for producing β-Ala-DOPA, comprising: Due to L-amino acid α-ligase activity, (1) A step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates. A method for producing β-Ala-DOPA, comprising:

[0014] Section 2. A method for producing β-Ala-DOPA, comprising: Using a microbial strain expressing L-amino acid α-ligase activity, (1) A step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates. Item 1. A method for producing β-Ala-DOPA according to Item 1, comprising:

[0015] Section 3. Item 3. The method for producing β-Ala-DOPA according to Item 2, wherein the microbial strain is a peptidase-deficient strain.

[0016] Section 4. A method for producing β-Ala-DOPA, comprising: Using a protein with L-amino acid α-ligase activity, (1) synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates in the presence of adenosine triphosphate (ATP); Item 1. A method for producing β-Ala-DOPA according to Item 1, comprising:

[0017] Section 5. 5. The method for producing β-Ala-DOPA according to any one of items 1 to 4, which is carried out in the presence of an antioxidant.

[0018] Section 6. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using L-amino acid α-ligase activity; and (2) a step of hydroxylating β-Ala-Tyr obtained in the step (1) by the activity of a two-component flavin-dependent monooxygenase to synthesize β-Ala-DOPA; A method for producing β-Ala-DOPA, comprising:

[0019] Section 7. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using a microbial strain expressing L-amino acid α-ligase activity; and (2) a step of hydroxylating β-Ala-Tyr obtained in the step (1) to synthesize β-Ala-DOPA using a microbial strain expressing two-component flavin-dependent monooxygenase activity; Item 7. The method for producing β-Ala-DOPA according to Item 6, comprising:

[0020] Section 8. 8. The method for producing β-Ala-DOPA according to Item 7, wherein the microbial strain is a peptidase-deficient strain.

[0021] Section 9. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) using a protein having L-amino acid α-ligase activity; and (2) a step of synthesizing β-Ala-DOPA by hydroxylating β-Ala-Tyr obtained in the step (1) in the presence of reduced nicotinamide adenine dinucleotide (NADH) using a protein having two-component flavin-dependent monooxygenase activity; Item 7. The method for producing β-Ala-DOPA according to Item 6, comprising:

[0022] Section 10. 10. The method for producing β-Ala-DOPA according to any one of items 6 to 9, which is carried out in the presence of an antioxidant. [Effects of the Invention]

[0023] The present invention makes it possible to efficiently synthesize β-Ala-DOPA. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows the plasmid map for producing tyrosine-containing dipeptide (β-Ala-Tyr). Vector: pET-21a(+), insert: ywfE (L-amino acid α-ligase derived from Bacillus subtilis ATCC 15245), primary sequence of expressed enzyme: YwfE modified enzyme N108A (C-terminal His-tag). [Figure 2] Figure 2 shows the plasmid map for producing DOPA-containing dipeptide (β-Ala-DOPA). Vector: pETDuet-1, insert: hpaBC (Pseudomonas aeruginosa PAO1-derived two-component flavin-dependent monooxygenase), primary sequence of expressed enzyme: co-expression of HpaB and HpaC. [Figure 3] FIG. 3 shows the antioxidant effect of ascorbic acid (5 mM to 20 mM) in the one-step production of β-Ala-DOPA by an enzymatic reaction system. [Figure 4] FIG. 4 shows the effect of using a PepD-deficient mutant strain on the one-step production of β-Ala-DOPA using a bacterial cell reaction system. [Figure 5] FIG. 5 shows the antioxidant effect of ascorbic acid in the one-step production of β-Ala-DOPA using a bacterial reaction system. DETAILED DESCRIPTION OF THE INVENTION

[0025] The method for producing β-Ala-DOPA of the present invention will be described in detail below.

[0026] In this specification, the terms "comprise" and "contain" are concepts that encompass all of "comprise," "consist essentially of," and "consist only of."

[0027] In this specification, when a numerical range is expressed as "A to B," it means A or more and B or less.

[0028] Herein, the amino acid sequence of a protein is described using the three-letter or single-letter notation of amino acids, which is well known and commonly used by those skilled in the art. Herein, amino acids are in the L-form unless otherwise specified. Herein, when describing a mutant protein, the single-letter notation of the amino acid into which a mutation is introduced in a wild-type protein, a number representing the mutation position, and the single-letter notation of the mutated amino acid are used, which is a method well known and commonly used by those skilled in the art.

[0029] As used herein, "a protein in which the amino acid residue corresponding to the Z1 residue (Z1) at the Yth position from the N-terminus of SEQ ID NO: X has been replaced with the Z2 residue (Z2)" refers to a protein having an amino acid sequence in which the Z1 residue at the Yth position from the N-terminus of the amino acid sequence of SEQ ID NO: X has been replaced with the Z2 residue when the amino acid sequence of the protein is aligned with SEQ ID NO: X so as to give the highest homology score.

[0030] "X" and "Y" represent integers of 1 or greater, "Z1" and "Z2" represent any amino acid, and "(Z1)" and "(Z2)" are the single-letter codes for each amino acid.

[0031] [1] One-step method for producing β-Ala-DOPA The method for producing β-Ala-DOPA of the present invention includes (1) the step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates using L-amino acid α-ligase activity. The present invention is a technology for synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) in one step in a bacterial reaction system or an enzymatic reaction system using a protein having L-amino acid α-ligase activity.

[0032] The one-step reaction from L-dopa is highly efficient.

[0033] [1-1] One-step method for producing β-Ala-DOPA using bacterial cells The method for producing β-Ala-DOPA of the present invention includes the steps of (1) synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates using a microbial strain that expresses L-amino acid α-ligase activity.

[0034] [ka]

[0035] In the method for producing β-Ala-DOPA of the present invention, the microbial strain is preferably a peptidase-deficient strain (transformed strain).

[0036] [1-2] One-step enzymatic reaction method for producing β-Ala-DOPA The method for producing β-Ala-DOPA of the present invention includes the steps of (1) synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates in the presence of adenosine triphosphate (ATP) using a protein having L-amino acid α-ligase activity.

[0037] [1-3] Proteins with L-amino acid α-ligase activity The L-amino acid α-ligase activity possessed by the protein of the present invention is, for example, imidazole dipeptide synthesis activity.

[0038] The protein having L-amino acid α-ligase activity is preferably a protein having L-amino acid α-ligase activity derived from Bacillus subtilis, a bacterium of the genus Bacillus (wild-type YwfE, SEQ ID NO: 1 and SEQ ID NO: 2 The protein having L-amino acid α-ligase activity may be a mutant YwfE of wild-type YwfE.

[0039] As a protein having the enzyme L-amino acid α-ligase activity, a commercially available enzyme provided by Kyowa Hakko Bio Co., Ltd. may be used.

[0040] The mutant YwfE (mutant enzyme, mutant protein) is preferably a mutant protein containing substitutions of at least 1 to 3 amino acid residues in the amino acid sequence of wild-type L-amino acid α-ligase: YwfE.

[0041] The mutant YwfE is a protein that has L-amino acid α-ligase activity and preferably has 80% or more homology with the wild-type amino acid sequence, more preferably 85% or more homology, even more preferably 90% or more homology, and particularly preferably 95% or more homology.

[0042] The mutant YwfE is preferably a protein having L-amino acid α-ligase activity, which consists of an amino acid sequence in which one or more amino acid residues of the wild-type amino acid sequence have been deleted, substituted, inserted, and / or added.

[0043] YwfE from the genus Bacillus is an L-amino acid α-ligase that recognizes many amino acids, including L-histidine, as C-terminal substrates and synthesizes dipeptides.

[0044] The present invention has revealed for the first time that wild-type YwfE and mutant YwfE have ligase activity for the N-terminal substrate β-alanine (β-Ala) and the C-terminal substrate L-dopa (L-DOPA), and are capable of synthesizing β-Ala-DOPA.

[0045] To evaluate L-amino acid α-ligase activity (dipeptide synthesis activity), for example, a test protein, an amino acid substrate, and adenosine triphosphate (ATP) are reacted in an aqueous buffer solution of pH 5 to pH 11 at a temperature of 20°C to 50°C for a predetermined time of 2 to 150 hours, and then the dipeptide synthesis activity is evaluated using high-performance liquid chromatography (HPLC) or the like.

[0046] The mutant YwfE is a mutant enzyme in which, for example, one, two (double mutant enzyme) or three (triple mutant enzyme) amino acid residues are substituted. The mutant YwfE has the amino acid sequence ( SEQ ID NO: 1and SEQ ID NO: 2 ) from the N-terminus, an enzyme in which the asparagine (N) residue at position 108 is substituted (mutated) with an alanine (A) residue (N108A) (YwfE, a mutant form of YwfE) can be used ( SEQ ID NO: 3 ).

[0047] [1-4] Protein-encoding nucleic acids The nucleic acid is obtained, for example, by introducing site-specific mutations using primers and the chromosomal DNA of a microorganism encoding the YwfE protein or a related protein thereof, such as Bacillus subtilis 168, as a template.

[0048] The desired mutations are introduced into the template gene using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as PCR or annealing (Muramatsu et al., eds., "New Genetic Engineering Handbook, Revised 4th Edition," Yodosha, pp. 82-88). If necessary, various commercially available site-directed mutagenesis kits, such as QuickChange II Site-Directed Mutagenesis Kit (Stratagene, USA) or QuickChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, USA), can be used.

[0049] Template DNA containing the YwfE gene is prepared by extracting genomic DNA from bacteria that produce the YwfE protein, or by extracting RNA and synthesizing cDNA by reverse transcription, according to a conventional method.

[0050] The bacterium that produces the YwfE protein is preferably a bacillus bacterium such as Bacillus subtilis. For example, the Bacillus sp. strains KSM-S237 (accession number FERM BP-7875), KSM-64 (accession number FERM BP-2886), and KSM-635 (accession number FERM BP-1485) are available at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (6 Central, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan).

[0051] Genomic DNA from Bacillus bacteria can be prepared, for example, by the method described in Pitcher et al., Lett. Appl. Microbiol., 1989, 8: pp. 151-156. Template DNA containing the YwfE gene is prepared by inserting a DNA fragment containing the YwfE gene excised from the prepared cDNA or genomic DNA into a vector of your choice.

[0052] Site-specific mutagenesis of the YwfE gene is most commonly achieved using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer is designed to anneal to a region of the YwfE gene containing a nucleotide sequence encoding the target amino acid residue and to contain a base sequence having a nucleotide sequence (codon) encoding the substituted amino acid residue in place of the nucleotide sequence (codon) encoding the target amino acid residue.

[0053] Using the nucleic acid obtained by the above method, a vector or plasmid is prepared for expressing wild-type YwfE or a mutant YwfE enzyme in a host strain, and a host strain into which this vector or plasmid has been introduced, i.e., a transformed strain, is produced, and wild-type YwfE or a mutant YwfE enzyme is then produced.

[0054] Expression vectors for preparing recombinant DNA are commercially available. These vectors can be used to prepare nucleic acid insertion vectors, which can then be used to prepare host cells containing the nucleic acid insertion vector, i.e., transformed cells.

[0055] When Escherichia coli is used as the host cell, preferred vectors include pColdI (Takara Bio), pCDF-1b, pRSF-1b (Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Biosciences), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), pET-3 (Novagen), pBluescriptII SK(+), pBluescript II KS(-) (Stratagene), and pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (accession number FERM BP-5407)].

[0056] When using the vector, any promoter may be used as long as it functions in host cells such as Escherichia coli, etc. Preferably, the promoter used is a promoter derived from Escherichia coli or a phage, such as the trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, or PSE promoter.

[0057] When the above vector is used, if a microorganism belonging to the genus Bacillus is used as the parent strain, the promoter is preferably an SPO1 promoter, an SPO2 promoter, a penP promoter, or the like that functions in Bacillus subtilis.

[0058] The promoter is preferably an artificially designed and modified promoter such as a promoter in which two Ptrp genes are connected in tandem, a tac promoter, a lacT7 promoter, or a let I promoter.

[0059] When a vector is used for the purpose of producing a protein having L-amino acid α-ligase activity, an expression vector is particularly useful. There are no particular limitations on the expression vector, as long as it expresses a protein in a test tube, in E. coli, in cultured cells, or in an individual organism. For in vitro expression, the expression vector is preferably the pBEST vector (manufactured by Promega). For E. coli, the expression vector is preferably the pET vector (manufactured by Invitrogen). For cultured cells, the expression vector is preferably the pME18S-FL3 vector (GenBank Accession No. AB009864). For individual organisms, the expression vector is preferably the pME18S vector (Mol Cell Biol. 8: 466-472 (1988)).

[0060] DNA can be inserted into a vector by a standard method, for example, by ligation using a restriction enzyme site. Reference: Current Protocols in Molecular Biology, Edited by Ausubel et al. (1987), Published by John Wiley & Sons, Sections 11.4-11.11

[0061] [1-5] Host strain The nucleic acid insertion vector is introduced into a host strain to obtain a transformed strain that expresses a protein having L-amino acid α-ligase activity.

[0062] The host strain is preferably a peptidase D deficient strain.

[0063] The host strain is preferably a yeast, a fungus, an alga, an animal cell, an insect cell, a bacterium, a plant cell, an archaea, or the like.

[0064] The host strain is preferably a yeast belonging to the genus Saccharomyces, Pichia, Kluyveromyces, Candida, Schizosaccharomyces, Debaryomyces, Yarrowia, Cryptococcus, Xanthophyllomyces, or the like. Examples of host strains that can be used include Saccharomyces cerevisiae, Saccharomyces bayanus, Pichia pastoris, Kluyveromyces lactis, Candida utilis, Candida glabrata, Schizosaccharomyces pombe, Debaryomyces hansenii, Yarrowia lipolytica, Cryptococcus curvatus, and Xanthophyllomyces dendrorhous.

[0065] The host strain is preferably a bacterium belonging to the genus Escherichia, Actinomycetes, Bacillus, Serratia, Pseudomonas, Corynebacterium, Brevibacterium, Rhodococcus, Lactobacillus, Streptomyces, Thermus, Streptococcus, or the like. Host strains include, for example, Escherichia coli, Bacillus subtilis, Bacillus brevis, Bacillus stearothermophilus, Serratia marcescens, Pseudomonas putida, Pseudomonas aeruginosa, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Rhodococcus erythropolis, and the like. erythropolis, Thermus thermophilus, Streptococcus lactis, Lactobacillus casei, Streptomyces lividans, etc. are used.

[0066] The host strain is preferably a filamentous fungus (mold) belonging to Aspergillus nigar, Aspergillus oryzae, or the like.

[0067] The host strain is preferably a microorganism belonging to the genus Mortierella, Fusarium, Schizochytrium, Thraustochytrium, etc. Examples of host strains that can be used include Mortierella ramanniana, Mortierella bainieri, Mortierella alpina, Cunninghamella elegans, Fusarium fujikuroi, Schizochytrium limacium, and Thraustochytrium aureum.

[0068] Introduction of the nucleic acid insertion vector into the host strain can be accomplished by a variety of methods, including calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene mediated transfection, protoplast fusion, liposome-mediated transfection (lipofection), conjugation, natural transformation, electroporation, and other methods known to those skilled in the art.

[0069] The expression vector is introduced into the host strain using commercially available transfection reagents. Reference: Current Protocols in Molecular Biology, 3 vols. Edited by Ausubel FM et al., John Wiley & Son, Inc., Current Protocols.

[0070] [1-6] Manufacturing method of β-Ala-DOPA [1-6-1] One-step method for producing β-Ala-DOPA using a bacterial reaction system In the method for producing β-Ala-DOPA of the present invention, a microbial strain (transformant) expressing a protein with L-amino acid α-ligase activity (wild-type YwfE or a YwfE mutant YwfE) is used and cultured in a culture medium, whereby β-Ala-DOPA can be synthesized from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates. The cultured cells are harvested, and the desired β-Ala-DOPA can be synthesized in a bacterial cell reaction system using the harvested cells as a biocatalyst. β-Ala-DOPA and L-dopa are added to the expression strain obtained by culturing to produce the desired β-Ala-DOPA, and β-Ala-DOPA is isolated from the reaction solution.

[0071] The produced β-Ala-DOPA is then isolated from the culture medium.

[0072] Of the components added to the culture medium, the carbon source is preferably one that can be assimilated by the microbial strain (transformant), and is preferably a carbohydrate such as glucose, fructose, sucrose, molasses containing these, starch, or starch hydrolysate, an organic acid such as acetic acid or propionic acid, or an alcohol such as ethanol or propanol.

[0073] Of the components added to the culture medium, preferred nitrogen sources include inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, ammonium salts of organic acids, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.

[0074] Among the components added to the culture medium, inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are preferably used.

[0075] Other components added to the cell culture medium preferably include nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acids, and various vitamins such as biotin.

[0076] Cultivation is usually carried out under aerobic conditions such as aeration and agitation, or shaking culture.

[0077] The culture temperature is not particularly limited as long as it is a temperature at which the microbial strain (transformant) can grow.

[0078] There are no particular limitations on the pH during cultivation, as long as the microbial strain (transformant) can grow at that pH. The pH during cultivation is adjusted by adding an acid or alkali.

[0079] Adding antioxidants When β-Ala-DOPA is synthesized from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates using a protein having L-amino acid α-ligase activity, an antioxidant (preferably ascorbic acid) is preferably added appropriately (approximately 5 mM to 50 mM).

[0080] The addition of an antioxidant (preferably ascorbic acid) primarily prevents the oxidation of L-dopa. The addition of an antioxidant can inhibit browning and polymerization of the substrate L-dopa, thereby suppressing the reduction of the substrate. The antioxidant can also inhibit the oxidative decomposition of the target compound, β-Ala-DOPA.

[0081] In any of the methods of the present invention, the addition of an antioxidant (such as ascorbic acid) makes it possible to stably produce the target product, β-Ala-DOPA (improving the production yield).

[0082] Lacking peptidase activity The present invention is the first to demonstrate that β-Ala-DOPA is degraded by peptidase D (pepD). Therefore, in the production method using the bacterial cells of the present invention, the degradation of β-Ala-DOPA can be suppressed by using a transformed strain lacking pepD. For example, by using Escherichia coli B series BL21(DE) as a host strain and deleting peptidase D (pepD), the degradation of β-Ala-DOPA can be suppressed, enabling highly efficient production of β-Ala-DOPA.

[0083] One-step production method of β-Ala-DOPA using a bacterial reaction system β-Ala-DOPA is produced by the condensation reaction of β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates, catalyzed by a protein with L-amino acid α-ligase activity.

[0084] Therefore, β-Ala-DOPA can be produced and obtained by culturing a microbial strain (transformant) that expresses a protein with L-amino acid α-ligase activity in a culture medium containing β-alanine (β-Ala) and L-dopa (L-DOPA), and then isolating and purifying the β-Ala-DOPA synthesized by adding the necessary substrates.

[0085] In the production method, the amounts of β-alanine (β-Ala) and L-dopa (L-DOPA) added are, for example, about 0.5 g / L to 100 g / L each.

[0086] The culture is usually carried out under aerobic conditions such as shaking culture or submerged aeration and stirring culture.

[0087] The culture temperature is preferably 15°C to 40°C.

[0088] The culture time is preferably 5 hours to 7 days.

[0089] The pH during the culture is preferably maintained at pH 3 to pH 9. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0090] To achieve a high-yield conversion reaction (synthesis of the target dipeptide β-Ala-DOPA from the substrates β-alanine (β-Ala) and L-dopa (L-DOPA)), it is preferable to harvest highly expressing cells and then react them in a reaction system (resting cell reaction).

[0091] The β-Ala-DOPA produced and accumulated in the buffer solution can be isolated and purified by methods commonly used by those skilled in the art, such as activated carbon, ion exchange resins, extraction with organic solvents, crystallization, thin layer chromatography, and high performance liquid chromatography.

[0092] In bacterial cell reactions using microbial strains (transformants), the harvested microbial strains (transformants) can be used as a biocatalyst in a buffer solution. For large-scale production on an industrial scale, it is preferable to control the pH during the reaction by lower limit control using alkali rather than a buffer solution.

[0093] [1-6-2] One-step enzyme reaction method for producing β-Ala-DOPA In the method for producing β-Ala-DOPA of the present invention, a protein having L-amino acid α-ligase activity (wild-type YwfE or mutant YwfE) is used, and by reacting it in a buffer solution in the presence of adenosine triphosphate (ATP), β-Ala-DOPA can be synthesized from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates.

[0094] The produced β-Ala-DOPA is then isolated from the buffer solution.

[0095] The buffer solution is preferably a phosphate buffer solution, a borate buffer solution, a citrate buffer solution, an acetate buffer solution, a Tris-HCl buffer solution, or the like, which are commonly used by those skilled in the art.

[0096] In the enzymatic reaction system production method, adenosine triphosphate (ATP) is used.

[0097] One-step enzymatic reaction method for producing β-Ala-DOPA β-Ala-DOPA is synthesized by the condensation reaction of β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates, catalyzed by a protein with L-amino acid α-ligase activity.

[0098] Therefore, β-Ala-DOPA can be produced and obtained by adding a protein with L-amino acid α-ligase activity to a buffer solution containing β-alanine (β-Ala) and L-dopa (L-DOPA), allowing the reaction to proceed for a predetermined period of time, and then isolating and purifying the synthesized β-Ala-DOPA.

[0099] In the production method, the amounts of β-alanine (β-Ala) and L-dopa (L-DOPA) added are, for example, about 0.5 g / L to 100 g / L each.

[0100] Adding antioxidants When synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates using a protein having L-amino acid α-ligase activity, an antioxidant (preferably ascorbic acid at an appropriate concentration (approximately 5 mM to 50 mM)) is preferably added.

[0101] In a production method using an enzymatic reaction system, an equivalent amount or more of adenosine triphosphate (ATP) is added to the substrate.

[0102] The enzymatic synthesis reaction is carried out in an aqueous medium, preferably at a pH of 5 to 11, and more preferably at a pH of 6 to 10.

[0103] The enzymatic synthesis reaction is carried out in an aqueous medium, preferably at 20°C to 50°C, more preferably at 25°C to 45°C.

[0104] The enzymatic synthesis reaction is carried out in an aqueous medium, preferably for 2 to 72 hours, more preferably for 6 to 36 hours.

[0105] The β-Ala-DOPA produced and accumulated in the buffer solution can be isolated and purified by methods commonly used by those skilled in the art, such as activated carbon, ion exchange resins, extraction with organic solvents, crystallization, thin layer chromatography, and high performance liquid chromatography.

[0106] [2] Two-step method for producing β-Ala-DOPA The method for producing β-Ala-DOPA of the present invention comprises the steps of (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using L-amino acid α-ligase activity, and (2) hydroxylating the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA using two-component flavin-dependent monooxygenase activity. The present invention is a technology for synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) in two steps in a bacterial or enzymatic reaction system: (1) synthesizing β-Ala-Tyr from β-Ala and L-tyrosine using a protein with L-amino acid α-ligase activity, and (2) synthesizing β-Ala-DOPA from β-Ala-Tyr using a protein with two-component flavin-dependent monooxygenase activity.

[0107] In terms of raw material costs, the two-step reaction from L-tyrosine (L-Tyr) is by far the most inexpensive method for producing β-Ala-DOPA. By converting L-tyrosine (L-Tyr) into a dipeptide and then converting the tyrosine (Tyr) into dopa (DOPA), it is possible to efficiently produce β-Ala-DOPA without going through unstable dopa (free DOPA).

[0108] [2-1] A two-step method for producing β-Ala-DOPA in a bacterial system (in vivo) The method for producing β-Ala-DOPA of the present invention comprises the steps of: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using a microbial strain (transformant) that expresses L-amino acid α-ligase activity; and (2) hydroxylating the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA using a microbial strain (transformant) that expresses two-component flavin-dependent monooxygenase activity.

[0109] [ka]

[0110] [ka]

[0111] In the method for producing β-Ala-DOPA of the present invention, the microbial strain is preferably a peptidase-deficient strain (transformed strain). When a bacterial cell reaction system is used in the two steps, the target of PepD is the final product β-Ala-DOPA, and it is therefore preferable to use a peptidase-deficient strain as a common transformant.

[0112] [2-2] Two-step enzymatic reaction method for producing β-Ala-DOPA The method for producing β-Ala-DOPA of the present invention comprises the steps of: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) using a protein having L-amino acid α-ligase activity; and (2) hydroxylating (introducing a hydroxyl group) the β-Ala-Tyr obtained in step (1) in the presence of reduced nicotinamide adenine dinucleotide (NADH) using a protein having two-component flavin-dependent monooxygenase activity to synthesize β-Ala-DOPA.

[0113] [2-3] Proteins with L-amino acid α-ligase activity [2-3-1] Protein In the method for producing β-Ala-DOPA of the present invention, in step (1), a microbial strain (transformant) expressing a protein having L-amino acid α-ligase activity (wild-type YwfE or a mutant form of YwfE) is used and cultured in a culture medium to prepare the protein, and β-Ala-Tyr is synthesized from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as reaction substrates in the presence of adenosine triphosphate (ATP).

[0114] The L-amino acid α-ligase activity of the protein of the present invention is as explained above.

[0115] Both wild-type and mutant YwfE possess ligase activity for the N-terminal substrate β-alanine (β-Ala) and the C-terminal substrate L-tyrosine (L-Tyr), and are capable of synthesizing β-Ala-Tyr.

[0116] Wild-type YwfE or a mutant form of YwfE is used as an enzyme to synthesize β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP).

[0117] [2-3-2] Protein-encoding nucleic acids The nucleic acid encoding the protein having L-amino acid α-ligase activity of the present invention is as explained above.

[0118] [2-3-3] Nucleic acid insertion vector The nucleic acid insertion vector containing the gene encoding the protein having L-amino acid α-ligase activity of the present invention and an appropriate expression sequence is as described above.

[0119] [2-3-4] Host strain into which the nucleic acid insertion vector is introduced The nucleic acid insertion vector of the present invention is introduced into a host strain, and the transformed strain that expresses a protein having L-amino acid α-ligase activity is as described above.

[0120] [2-4] Proteins with two-component flavin-dependent monooxygenase activity The protein with two-component flavin-dependent monooxygenase activity (HpaBC) is a monooxygenase composed of two components: a flavin-dependent hydroxylase (HpaB: 4-hydroxyphenylacetate (4HPA) 3-monooxygenase) and a protein with flavin reductase activity (HpaC: NAD(P)H-flavin oxidoreductase).

[0121] SEQ ID NO: 4: HpaB amino acid sequence HpaB:Pseudomonas aeruginosa PA01, -HpaB(520aa) SEQ ID NO: 5: HpaB (1,563 bp) SEQ ID NO: 8: HpaC amino acid sequence HpaC:Pseudomonas aeruginosa PA01, -HpaC(179aa) SEQ ID NO: 9: HpaC (513 bp)

[0122] In the method for producing β-Ala-DOPA of the present invention, in step (2), a microbial strain (transformant) expressing a protein having two-component flavin-dependent monooxygenase activity (preferably HpaBC, EC: 1.14.14.9.) is used and cultured in a culture medium to hydroxylate (introduce a hydroxyl group into an -OH group) the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA.

[0123] [2-5] Manufacturing method of β-Ala-DOPA The two-step bacterial process for producing β-Ala-DOPA involves: (1) Using a microbial strain (transformant) that expresses L-amino acid α-ligase activity, a process is carried out to synthesize β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates; Next, (2) a microbial strain (transformant) expressing two-component flavin-dependent monooxygenase activity may be used to hydroxylate the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA.

[0124] [2-5-1] Two-step method for producing β-Ala-DOPA using a bacterial reaction system Step (1): synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates In step (1), a microbial strain (transformant) expressing a protein with L-amino acid α-ligase activity (wild-type YwfE or a mutant form of YwfE) is used to synthesize β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates.

[0125] In step (1), a microbial strain (transformant) expressing a protein having L-amino acid α-ligase activity is cultured in a culture medium, and then β-Ala-Tyr synthesized in a reaction solution containing β-alanine (β-Ala) and L-tyrosine (L-Tyr) is isolated and purified to produce and obtain β-Ala-Tyr.

[0126] In the production method, the amounts of β-alanine (β-Ala) and L-tyrosine (L-Tyr) added are, for example, about 0.5 g / L to 100 g / L each.

[0127] To achieve a high-yield conversion reaction (synthesis of the target dipeptide β-Ala-Tyr from the substrates β-alanine (β-Ala) and L-tyrosine (L-Tyr)), it is preferable to harvest highly expressing cells and then react them in a reaction system (resting cell reaction).

[0128] Step (2): A step of synthesizing β-Ala-DOPA by hydroxylating β-Ala-Tyr obtained in the step (1). In step (2), a microbial strain (transformant) that expresses a protein with two-component flavin-dependent monooxygenase activity (preferably HpaBC, a complex enzyme consisting of HpaB (EC 1.14.14.9) and HpaC (flavin reductase: EC 1.5.1.36)) is used and cultured in a culture medium to hydroxylate (introduce a hydroxyl group) the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA.

[0129] As HpaBC, HpaBC derived from bacteria of the genus Escherichia, HpaBC derived from bacteria of the genus Pseudomonas, and the like can be used.

[0130] In step (2), β-Ala-DOPA is produced by culturing a microbial strain (transformant) expressing a protein having two-component flavin-dependent monooxygenase activity in a culture medium containing β-Ala-Tyr. The synthesized β-Ala-DOPA may be isolated and purified.

[0131] The culture is usually carried out under aerobic conditions such as shaking culture or submerged aeration and stirring culture.

[0132] The culture temperature is preferably 15°C to 40°C.

[0133] The culture time is preferably 5 hours to 7 days.

[0134] The pH during the culture is preferably maintained at pH 3 to pH 9. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0135] To achieve a high-yield conversion reaction (synthesis of the target dipeptide, β-Ala-DOPA, from the substrate β-Ala-Tyr), it is preferable to harvest highly expressing cells and then react them in a reaction system (resting cell reaction).

[0136] Adding antioxidants When synthesizing β-Ala-DOPA, it is preferable to add an antioxidant (preferably ascorbic acid) appropriately (about 5 mM to 50 mM). By adding an antioxidant (preferably ascorbic acid), it is possible to suppress oxidative decomposition of the target compound, β-Ala-DOPA.

[0137] The β-Ala-DOPA produced and accumulated in the buffer solution can be isolated and purified by methods commonly used by those skilled in the art, such as activated carbon, ion exchange resins, extraction with organic solvents, crystallization, thin layer chromatography, and high performance liquid chromatography.

[0138] In the bacterial cell reaction system, the harvested microbial strain (transformed strain) can be used as a biocatalyst to react in a buffer solution. For large-scale industrial production, it is preferable to control the pH during the reaction by controlling the lower limit with alkali rather than a buffer solution.

[0139] [2-5-2] Two-step enzymatic reaction method for producing β-Ala-DOPA Step (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) using a protein having L-amino acid α-ligase activity. , In step (1), a protein with L-amino acid α-ligase activity (wild-type YwfE or a mutant YwfE) is used to synthesize β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in a buffer solution in the presence of adenosine triphosphate (ATP).

[0140] In a production method using an enzymatic reaction system, an equivalent amount or more of adenosine triphosphate (ATP) is added to the substrate.

[0141] In step (1), β-Ala-Tyr is synthesized by a condensation reaction of β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) under the catalytic action of a protein with L-amino acid α-ligase activity.

[0142] In the production method, the amounts of β-alanine (β-Ala) and L-tyrosine (L-Tyr) added are, for example, about 0.5 g / L to 100 g / L each.

[0143] In step (1), a protein having L-amino acid α-ligase activity is added to a buffer solution containing β-alanine (β-Ala) and L-tyrosine (L-Tyr), and after incubation for a predetermined period of time, the synthesized β-Ala-Tyr is isolated and purified, thereby producing and obtaining β-Ala-Tyr.

[0144] Step (2): Using a protein having two-component flavin-dependent monooxygenase activity, β-Ala-Tyr obtained in step (1) is hydroxylated in the presence of reduced nicotinamide adenine dinucleotide (NADH) to synthesize β-Ala-DOPA. In step (2), a protein having two-component flavin-dependent monooxygenase activity (preferably HpaBC) is used to synthesize β-Ala-DOPA in the presence of nicotinamide adenine dinucleotide (NADH) by hydroxylation of β-Ala-Tyr obtained in step (1). In step (2), reduced nicotinamide adenine dinucleotide (NADH) is added.

[0145] In the production method using an enzymatic reaction system, an equivalent or greater amount of reduced nicotinamide adenine dinucleotide (NADH) is added to the substrate.

[0146] In step (2), β-Ala-DOPA is synthesized by hydroxylation of β-Ala-Tyr as a substrate, catalyzed by a protein having two-component flavin-dependent monooxygenase activity.

[0147] In step (2), the amount of β-Ala-Tyr to be added is, for example, about 0.1 g / L to 100 g / L.

[0148] In step (2), a protein having two-component flavin-dependent monooxygenase activity is added to a buffer solution containing β-Ala-Tyr, and after incubation for a predetermined period of time, β-Ala-DOPA can be produced and obtained.

[0149] The synthesis reaction is carried out in an aqueous medium, preferably at a pH of 5 to 11, more preferably at a pH of 6 to 10.

[0150] The synthesis reaction is carried out in an aqueous medium, preferably at 20°C to 50°C, more preferably at 25°C to 45°C.

[0151] The synthesis reaction is carried out in an aqueous medium, preferably for 2 to 72 hours, more preferably for 6 to 36 hours.

[0152] Adding antioxidants When synthesizing β-Ala-DOPA, it is preferable to add an antioxidant (preferably ascorbic acid) appropriately (about 5 mM to 50 mM). By adding an antioxidant (preferably ascorbic acid), it is possible to suppress oxidative decomposition of the target compound, β-Ala-DOPA.

[0153] The β-Ala-DOPA produced and accumulated in the buffer solution can be isolated and purified by methods commonly used by those skilled in the art, such as activated carbon, ion exchange resins, extraction with organic solvents, crystallization, thin layer chromatography, and high performance liquid chromatography. [Example]

[0154] The examples illustrate embodiments of the invention and should not be construed as limiting the scope of the invention.

[0155] In the following examples, preparation of polynucleotides (DNA, mRNA), PCR, base sequencing, microbial strains (transformants), protein expression, protein purification, HPLC analysis, etc. are carried out using methods well known and commonly used by those skilled in the art.

[0156] See, for example, Sambrook, J. and Russell, DW, Molecular Cloning A Laboratory Manual 4th Edition, Cold Spring Harbor Laboratory Press (2012).

[0157] [1] Site-directed mutagenesis of YwfE YwfE gene ( SEQ ID NO: 1The desired mutations were introduced using Quick Change Site-Directed Mutagenesis (Strategene, USA) with a pET vector incorporating the fragment (1419 bp, 472 residues) as a template (Figure 1).

[0158] PCR reactions were carried out under the reaction conditions shown in Table 1 (composition) and Table 2 (PCR cycle). KOD-Plus-Neo-DNA polymerase (Toyobo Co., Ltd., Osaka) was used in the PCR reactions.

[0159] Using primers, a YwfE protein was generated in which the 108th asparagine (N) residue from the N-terminus of the YwfE protein was replaced with an alanine (A) residue ( SEQ ID NO: 3 A plasmid was obtained in which a site-specific mutation of polynucleotide N108A, which is involved in the nucleotide sequence of ...

[0160] [Table 1]

[0161] [Table 2]

[0162] After the PCR reaction, the resulting PCR product was analyzed using a DNA sequencer (Applied Biosystems, Life Technologies Japan, Tokyo) to confirm whether site-specific mutations had been introduced.

[0163] The vector extracted from E. coli has the Dpn I site methylated by Dam methylase, while the PCR product has the Dpn I site unmethylated. This allows us to distinguish between the template vector and the PCR product. Briefly, to remove the template vector from the reaction mixture after PCR, the purified PCR product was treated with Dpn I at 37°C for 2 hours.

[0164] The restriction enzyme reaction was carried out under the reaction conditions shown in Table 3.

[0165] [Table 3]

[0166] After digestion with the restriction enzyme Dpn I, the site-directed mutagenesis plasmid was purified by phenol-chloroform treatment and ethanol precipitation, and then dissolved in TE buffer, pH 8.0.

[0167] [2] Transformation of E. coli JM109 with site-directed mutagenesis plasmid Competent cells of E. coli JM109 and the site-directed mutagenesis plasmid were heat-treated at 42°C. After that, SOC medium was added and cultured, and the cells were inoculated onto LB agar medium containing 50 μg / mL of kanamycin and cultured overnight at 37°C.

[0168] One colony was selected from the grown colonies, suspended in 3 mL of kanamycin-containing LB medium, and cultured at 37°C for 5 hours. After cultivation, the site-directed mutagenesis plasmid was extracted from the transformed strain using the alkaline SDS method. The extracted site-directed mutagenesis plasmid was purified by phenol-chloroform treatment and ethanol precipitation, and then dissolved in TE buffer (pH 8.0).

[0169] [3] Preparation of purified enzyme Transformation of Escherichia coli BL21 with site-directed mutagenesis plasmids The purified site-directed mutagenesis plasmid was used to transform E. coli BL21 by the heat shock method. Competent E. coli BL21 cells and the site-directed mutagenesis plasmid were both heat-treated at 42°C. The cells were then cultured in SOC medium, inoculated onto LB agar medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C.

[0170] The E. coli that formed colonies was designated as a YwfE-expressing transformant strain.

[0171] Induction of expression by IPTG One colony was selected from the grown colonies of the YwfE-expressing transformant and suspended in 3 mL of kanamycin-containing LB medium in a test tube. The colony was pre-cultured at 37°C for 5 hours. Next, 2 mL of the pre-culture was added to 200 mL of kanamycin-containing LB medium in a 500 mL baffled Erlenmeyer flask. After 2 hours of main culture at 37°C and 120 rpm, 200 μL of 100 mM IPTG was added. The IPTG-added culture was then cultured overnight at 25°C and 120 rpm.

[0172] After overnight culture, the cells were collected by centrifugation at 5,000 × g for 10 minutes, and the cell pellet was washed by suspending it in 100 mM Tris-HCl buffer (pH 8.0). This procedure was repeated twice to remove medium components. After washing, the cell pellet was frozen and stored at -80°C.

[0173] Enzyme purification BugBuster™ Protein Extraction Reagent (Merck KGaA, Germany) and lysozyme were added to the thawed bacterial pellet to extract proteins. The bacterial lysate was separated into a precipitate (insoluble fraction) and a supernatant (cell-free extract) by centrifugation. The cell-free extract was applied using a His GraviTrap (GE Healthcare, USA). The eluted solution was desalted using a PD-10 column (GE Healthcare).

[0174] Measurement of purified enzyme concentration The concentration of the purified enzyme was measured using a microplate reader MODEL 550 (Bio-Rad, USA) after a color reaction using Coomassie Brilliant Blue. The concentration of the purified enzyme was determined from the absorbance at 595 nm using the calibration curve method.

[0175] [4] β-Ala-DOPA or β-Ala-Tyr synthesis activity evaluation (HPLC analysis) Standards of β-Ala-DOPA or β-Ala-Tyr were used. The amounts of β-Ala-DOPA or β-Ala-Tyr synthesized were determined by HPLC analysis using Nα-(5-fluoro-2,4-dinitrophenyl)-L-alanineamide (FDAA) derivatization and quantified using a calibration curve. HPLC analysis was performed according to the standard method under the conditions shown in Table 4 (eluent composition) and Table 5 (gradient program).

[0176] <Analysis conditions> Equipment used: HITACHI L-7000 series (Hitachi, Ltd., Tokyo) Column used: WH-C18A (4 × 150 mm) (Hitachi High-Technologies Corporation, Tokyo) Sample injection volume: 10 μL Flow rate: 0.5mL / min Column temperature: 40℃ UV detection wavelength: 340 nm

[0177] [Table 4]

[0178] [Table 5]

[0179] The enzyme was the wild-type enzyme (YwfE) ( SEQ ID NO: 2 ), and the YwfE single mutant enzyme N108A( SEQ ID NO: 3 ) was used.

[0180] After the reaction was completed, β-Ala-DOPA or β-Ala-Tyr was analyzed by HPLC.

[0181] Statistical tests are performed using the statistical testing software EZR (http: / / www.jichi.ac.jp / saitama-sct / SaitamaHP.files / statmed.htm) The concentrations of β-Ala-DOPA or β-Ala-Tyr produced by the wild-type enzyme and by each mutant enzyme were analyzed post hoc by one-way analysis of variance (ANOVA) and Dunnett's test with a p-value of 0.05 as the significance level (n = 3 per group).

[0182] [5] One-step production of β-Ala-DOPA The one-step production of β-Ala-DOPA using a bacterial reaction system involves (1) synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates using host cells expressing L-amino acid α-ligase activity (wild-type YwfE or a YwfE mutant YwfE).

[0183] The host strain is preferably a peptidase-deficient strain.

[0184] In a bacterial cell reaction system using a host strain (Escherichia coli), ATP-free synthesis is possible by supplying sugars. In this bacterial cell reaction system, β-Ala-DOPA can be synthesized in high yield from β-Ala and DOPA by using a host strain with a defective mutation in the host's peptidase (PepD).

[0185] The one-step production of β-Ala-DOPA using an enzymatic reaction system involves (1) synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates in the presence of adenosine triphosphate (ATP) using a protein with L-amino acid α-ligase activity (YwfE, wild-type or its mutant enzyme).

[0186] The enzymatic one-step production of β-Ala-DOPA is preferably carried out in the presence of an antioxidant (preferably ascorbic acid (10 mM)). L-DOPA is prone to browning and polymerization due to oxidative decomposition. When L-DOPA is used as a substrate, the addition of an antioxidant (preferably ascorbic acid (approximately 10 mM to 20 mM)) can inhibit the browning and polymerization of L-DOPA, thereby maintaining L-DOPA stability and preventing its depletion.

[0187] [5-1] One-step production of β-Ala-DOPA using an enzymatic reaction system Antioxidant effect of ascorbic acid In the enzyme system, by adding ascorbic acid at concentrations of 5 mM to 20 mM, the desired product β-Ala-DOPA was synthesized from the substrate L-dopa (L-DOPA) with a conversion rate of over 90% (Figure 3).

[0188] [Table 6]

[0189] [5-2] One-step production of β-Ala-DOPA using a bacterial reaction system Effect of using PepD-deficient mutant strain In a bacterial system using a recombinant E. coli strain, the addition of a small amount of sugar makes it possible to create a reaction system without the addition of ATP. In a bacterial reaction system using a recombinant E. coli strain, a host with a deletion mutation of peptidase (PepD) is preferably used.

[0190] In the bacterial cell reaction system, by using a recombinant Escherichia coli strain with a deletion mutation of peptidase (PepD), we were able to synthesize the target product β-Ala-DOPA from the substrate L-dopa (L-DOPA) with a conversion rate of over 50% (Figure 4).

[0191] [Table 7]

[0192] Effect of increasing the amount of bacterial cells used In the bacterial system, by increasing the bacterial mass, it was possible to synthesize the target product β-Ala-DOPA from the substrate L-dopa with a conversion rate of over 70%.

[0193] [Table 8]

[0194] Antioxidant effect of ascorbic acid In the bacterial cell reaction system, by adding ascorbic acid at concentrations of 5mM to 20mM, the target product β-Ala-DOPA could be synthesized from the substrate L-dopa (L-DOPA) with a conversion rate of over 60% (Figure 5). Furthermore, by optimizing the reaction conditions, such as increasing the amount of bacterial cells, it is possible to ultimately increase the yield (conversion rate) to approximately 76%.

[0195] [Table 9]

[0196] [6] Two-step production of β-Ala-DOPA The two-step production of β-Ala-DOPA using a bacterial reaction system involves the following steps: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using a strain co-expressing L-amino acid α-ligase activity (wild-type YwfE or a mutant form of YwfE) and two-component flavin-dependent monooxygenase activity; and (2) hydroxylating (introducing a hydroxyl group) the β-Ala-Tyr obtained in step (1) to synthesize β-Ala-DOPA.

[0197] β-Ala-Tyr can be produced by adding β-Ala and Tyr as substrates to a microbial strain (transformant) that expresses L-amino acid α-ligase activity. β-Ala-Tyr can be produced by adding β-Ala-Tyr as a substrate to a microbial strain (transformant) that expresses a two-component flavin-dependent monooxygenase. The two-component flavin-dependent monooxygenase and the strain that expresses it are as described above. By using a bacterium that simultaneously expresses two enzymes, L-amino acid α-ligase activity (YwfE) and two-component flavin-dependent monooxygenase (HaBC), a two-step reaction can be carried out simultaneously.

[0198] The microbial strain is preferably a peptidase-deficient strain (transformed strain).

[0199] In a bacterial cell reaction system using a microbial strain (such as Escherichia coli), adding a small amount of sugar makes it possible to create an ATP-free system. In a bacterial system, by using a transformant with a defective mutation in the bacterial peptidase (PepD), it is possible to synthesize β-Ala-DOPA from β-Ala and DOPA in high yield.

[0200] The two-step enzymatic reaction system for the production of β-Ala-DOPA involves the following steps: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) using a protein with L-amino acid α-ligase activity; and (2) hydroxylating the β-Ala-Tyr obtained in step (1) in the presence of reduced nicotinamide adenine dinucleotide (NADH) using a protein with two-component flavin-dependent monooxygenase activity to synthesize β-Ala-DOPA.

[0201] In step (1), the synthesis of dipeptides by the L-amino acid α-ligase activity (wild-type YwfE or mutant YwfE) requires ATP, so the enzyme reaction system must be supplied with ATP. In step (2), β-Ala-Tyr is hydroxylated and converted to β-Ala-DOPA using a two-component flavin-dependent monooxygenase (HpaBC). The enzyme reaction system must be supplied with NADH.

[0202] [6-1] Two-step production of β-Ala-DOPA using a bacterial reaction system Step (1) Synthesis of β-Ala-Tyr In step (1), β-Ala-Tyr was synthesized using a recombinant Escherichia coli strain expressing a site-directed mutant enzyme YwfE (N108A) of the enzyme YwfE, which has L-amino acid α-ligase activity (YwfE, dipeptide synthase).

[0203] For producing tyrosine-containing dipeptide (β-Ala-Tyr) Host: E. coli BL21 (DE3) Plasmid information Vector: pET-21a (+) Insert: ywfE (L-amino acid ligase from Bacillus subtilis ATCC 15245) Primary sequence of the expressed enzyme: YwfE modified enzyme N108A (C-terminal His-tag)

[0204] [Table 10]

[0205] Quantitative analysis by LC-MS confirmed that 4.73 mM of β-Ala-Tyr (target compound) was synthesized from 12.5 mM of β-Ala (substrate).

[0206] When the culture scale was increased from 300 μL to 3 mL, the synthesis yield was increased to 9.23 mM β-Ala-Tyr (target compound) (conversion rate 73.8%).

[0207] It was suggested that aeration and stirring conditions are important when changing the reaction vessel, and the use of a jar fermenter is preferable for scale-up.

[0208] Step (2) Synthesis of β-Ala-DOPA (method using Escherichia coli as a bacterial cell reaction system) Step (2) is the conversion process from β-Ala-Tyr to β-Ala-DOPA.

[0209] As a preliminary study, it was confirmed that saturated L-dopa aqueous solution, when left at 30℃ for 20 hours, turned brown at pH 6.5 or higher.

[0210] Using a recombinant Escherichia coli strain overexpressing a two-component flavin-dependent monooxygenase (HpaBC), β-Ala-Tyr was hydroxylated as a substrate to synthesize β-Ala-DOPA.

[0211] For producing DOPA-containing dipeptide (β-Ala-DOPA) Host: E. coli BL21 (DE3) Plasmid information Vector: pETDuet-1 Insert: hpaBC (two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa PAO1) Primary sequences of expressed enzymes: Co-expression of HpaB and HpaC Quantitation by LC-MS confirmed the reduction of β-Ala-Tyr (substrate) and the production of β-Ala-DOPA (target compound) (yield: 16.7%).

[0212] In the reaction system to which ascorbic acid was added, the reaction proceeded without browning under alkaline conditions of pH 8.0, and it is believed that L-dopa was successfully converted to β-alanine.

[0213] [Table 11]

[0214] SEQ ID NO: 1: Wild-type YwfE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ATG GAG AGA AAA ACA GTA TTG GTC ATC GCT GAT CTT GGA GGC TGC CCG CAC ATG TTT 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 TAT AAA AGC GCT GCT GAA AAA TAT AAC CTG GTC AGC TTT ATT CCA AGA CCT TTT GCA ATT 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACA GCC TCC CAT GCA GCA TTG ATT GAA AAA TAC TCG GTC GTC GTC ATA AAA GAT AAA GAC 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 TAT TTT AAG AGT TTA GCT GAT TTT GAA CAC CCT GAT TCC ATT TAT TGG GCG CAT GAA GAT 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 CAT AAC AAG CCT GAG GAA GAG GTC GTC GAG CAA ATC GTC AAG GTT GCC GAA ATG TTT GGG 101 102 103 104 105 106 107 108 109 110 111 112 GCG GAT GCC ATC ACA ACA AAC AAT GAA TTA TTC ATT GCT CCG ATG GCG AAA GCC TGT GAA 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 CGT CTG GGC TTG AGA GGT GCC GGC GTG CAG GCA GCC GAA AAT GCC AGA GAT AAA AAT AAA 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 ATG AGG GAC GCT TTT AAT AAG GCC GGA GTC AAA TCG ATC AAA AAC AAA CGA GTC ACA ACT 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 CTT GAA GAT TTC CGT GCT GCT CTT GAA GAG ATC GGC ACA CCT CTT ATC TTA AAG CCT ACA 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 TAC TTA GCG AGT TCT ATC GGT GTA ACG CTG ATT ACG GAC ACT GAG ACG GCA GAA GAT GAA 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 TTT AAC AGA GTC AAT GAC TAT CTG AAA TCA ATT AAC GTG CCA AAG GCG GTT ACG TTT GAA 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 GCG CCG TTT ATC GCT GAA GAA TTT TTA CAG GGT GAG TAC GGA GAC TGG TAT CAA ACA GAA 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 GGG TAC TCC GAC TAT ATC AGT ATA GAA GGC ATC ATG GCT GAC GGT GAG TAT TTC CCG ATC 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 GCC ATT CAT GAT AAA ACG CCG CAA ATC GGG TTT ACA GAG ACA TCC CAC ATT ACG CCG TCC 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 ATT CTG GAT GAA GAG GCA AAA AAG AAA ATT GTC GAA GCT GCC AAA AAG GCA AAT GAA GGG 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 CTT GGA CTG CAA AAT TGC GCA ACA CAT ACA GAG ATC AAG CTA ATG AAA AAC AGA GAA CCG 321 322 323 324 325 326 327 328 329 310 331 332 333 334 335 336 337 338 339 340 GGT TTA ATA GAG TCG GCA GCC AGA TTT GCC GGC TGG AAT ATG ATC CCC AAT ATT AAA AAG 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 GTC TTT GGC CTT GAT ATG GCG CAA TTA TTA TTA GAT GTC CTC TGT TTC GGA AAA GAC GCC 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 GAT CTG CCG GAC GGA TTA TTG GAT CAA GAG CCT TAT TAT GTT GCC GAC TGC CAT TTG TAC 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 CCG CAG CAT TTC AAA CAA AAT GGC CAA ATT CCT GAA ACT GCT GAG GAT TTG GTC ATT GAA 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 GCG ATC GAT ATT CCG GAC GGG CTT TTA AAA GGG GAT ACT GAA ATC GTT TCT TTT TCG GCC 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 GCA GCA CCA GGC ACT TCA GTT GAT TTG ACA TTG TTT GAA GCT TTC AAT TCC ATT GCT GCA 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 TTT GAA CTG AAA GGC AGT AAT TCA CAG GAT GTG GCT GAA TCA ATC AGA CAA ATT CAG CAG 461 462 463 464 465 466 467 468 469 470 471 472 END CAT GCG AAG CTG ACG GCA AAG TAT GTG CTG CCA GTA TGA

[0215] SEQ ID NO: 2: Amino acid sequence of wild-type YwfE 1 MERKTVLVIA DLGGCPPHMF YKSAAEKYNL VSFIPRPFAI TASHAALIEK 50 51 YSVAVIKDKD YFKSLADFEH PDSIYWAHED HNKPEEEVVE QIVKVAEMFG 101 ADAITTNNEL FIAPMAKACE RLGLRGAGVQ AAENARDKNK MRDAFNKAGV 150 151 KSIKNKRVTT LEDFRAALEE IGTPLILKPT YLASSIGVTL ITDTETAEDE 200 201 FNRVNDYLKS INVPKAVTFE APFIAEEFLQ GEYGDWYQTE GYSDYISIEG 250 251 IMADGEYFPI AIHDKTPQIG FTETSHITPS ILDEEAKKKI VEAAKKANEG 300 301 LGLQNCATHT EIKLMKNREP GLIESAARFA GWNMIPNIKK VFGLDMAQLL 350 351 LDVLCFGKDA DLPDGLLDQE PYYVADCHLY PQHFKQNGQI PETAEDLVIE 400. 401 AIDIPDGLLK GDTEIVSFSA AAPGTSVDLT LFEAFNSIA FELKGSNSQD 451 VAESIRQIQQ HAKLTAKYVL PV 472

[0216] SEQ ID NO: 3: Amino acid sequence of YwfE with N108A mutation introduced 1 MARKET TVLVIA DLGGCPPHMF YKSAAEKYNL VSFIPRPFAI TASHAALIEK 51 YSVAVIKDKD YFKSLADFEH PDSIYWAHED HNKPEEEVVE QIVKVAEMFG 101 ADAITTNAEL FIAPMAKACE RLGLRGAGVQ AAENARDKNK MRDAFNKAGV 150 151 KSIKNKRVTT LEDFRAALEE IGTPLILKPT YLASSIGVTL ITDTETAEDE 200 201 FNRVNDYLKS INVPKAVTFE APFIAEEFLQ GEYGDWYQTE GYSDYISIEG 250 251 IMADGEYFPI AIHDKTPQIG FTETSHITPS ILDEEAKKKI VEAAKKANEG 300 301 LGLQNCATHT EIKLMKNREP GLIESAARFA GWNMIPNICK VFGLDMAQLL 351 LDVLCFGKDA DLPDGLLDQE PYYVADCHLY PQHFKQNGQI PETAEDLVIE 400. 401 AIDIPDGLLK GDTEIVSFSA AAPGTSVDLT LFEAFNSIA FELKGSNSQD 451 VAESIRQIQQ HAKLTAKYVL PVLEHHHHHH 480

[0217] SEQ ID NO: 4: HpaB amino acid sequence 1 MKPEDFRASA TRPFTGEEYL ASLRDDREIY IYGDRVKDVT SHPAFRNAAA 50 51 SMARLYDALH DPQSKEKLCW ETDTGNGGYT HKFFRYARSA DELRQQRDAI 100 101 AEWSRLTYGW MGRTPDYKAA FGSALGANPG FYGRFEDNAK TWYKRIQEAC 150 151 LYLNHAIVNP PIDRDKPVDQ VKDVFISVDE EVDGGIVVSG AKVVATNSAL 200 201 THYNFVGQGS AQLLGDNTDF ALMFIAPMNT PGMKLICRPS YELVAGIAGS 250 251 PFDYPLSSRF DENDAILVMD KVFIPWENVL YRDFERCKQ WFPQGGFGRL 300 301 FPMQGCTRLA VKLDFITGAL YKALQCTGSL EFRGVQAQVG EVVAWRNLFW 350 351 SLTDAMYGNA SEWHGGAFLP SAEALQAYRV LAPQAYPEIK KTIEQVVASG 400 401 LIYLPSGVRD LHNPQLDKYL STYCRGSGGM GHRERIKILK LLWDAIGSEF 450 451 GGRHELYEIN YAGSQDEIRM QALRQAIGSG AMKGMLGMVE QCMGDYDENG 500 501 WTVPHLHNPD DINVLDRIRQ 520

[0218] SEQ ID NO: 5: HpaB atgaaacccg aagatttccg tgcctctgcc acccgtccgt tcaccggcga ggagtacctc gccagcctgc gcgacgaccg tgagatctac atctacggcg accgcgtcaa ggacgtcacc agccaccccg ccttccgcaa cgcggccgcc tccatggccc ggctctacga cgccctgcac gatccgcaga gcaaggaaaa gctctgctgg gagaccgata ccggcaacgg cggctatacc cacaagttct tccgctacgc gcgcagcgcc gacgaactgc gccagcagcg cgacgccatc gccgagtggt cgcggctgac ctacggctgg atgggccgca ccccggacta caaggccgcc ttcggcagcg ccctcggcgc caacccgggc ttctacgggc gtttcgagga caacgcgaaa acctggtaca agcgcatcca ggaagcctgc ctgtacctca accatgccat cgtcaacccg ccgatcgacc gcgacaagcc ggtggaccag gtcaaggacg tgttcatctc ggtggacgag gaagtcgacg gcggcatcgt cgtcagcggc gccaaggtgg tcgccacgaa ttccgcgctg acccactaca acttcgtcgg ccagggttcg gcgcaactgc tcggcgacaa caccgacttc gccctgatgt tcatcgcgcc gatgaacacc cccggcatga agctgatctg ccgcccctcc tacgaactgg tggcgggtat cgccggatcg ccgttcgact acccgctgtc cagccgtttc gacgagaacg acgcgatcct ggtgatggac aaggtgttca tcccctggga gaacgtactg atctaccgcg acttcgagcg ctgcaagcag tggttccccc agggtggctt cggccggctg ttcccgatgc agggctgcac ccgcctggcg gtcaagctcg acttcatcac cggcgccctc tacaaggccc tgcaatgcac cggctccctg gagttccgcg gcgtgcaggc gcaggtcggc gaagtggtgg cctggcgcaa cctgttctgg tcgctgaccg acgccatgta cggcaacgcc agcgaatggc acggcggcgc cttcctgccc agcgccgagg ccctgcaggc ctaccgcgtg ctggcgccgc aggcctaccc ggagatcaag aagaccatcg agcaggtggt cgccagcggc ctgatctacc tgccctccgg cgttcgcgac ctgcacaatc cgcaactcga caagtatctc Tccacctatt gccgcggctc cggcggcatg ggccaccggg agcggatcaa gatcctcaag ctgctctggg acgccatcgg cagcgagttc ggcggccgcc acgagctgta cgagatcaac tacgccggca gccaggacga gatccgcatg caggccctgc gccaggcgat cggcagcggg gcgatgaagg gcatgctcgg catggtcgag cagtgcatgg gcgactacga cgagaacggc tggaccgtgc cgcacctgca caacccggac gacatcaacg tgctcgatcg catccgccaa tga

[0219] SEQ ID NO: 6: HpaB primer (forward) forward : 5' -ttctcatgaaacccgaagatttccgtgcct-3’

[0220] SEQ ID NO: 7: HpaB primer (reverse) reverse :5’-cgcggatcctcattggcggatgcgatcgag-3’

[0221] SEQ ID NO: 8: HpaC amino acid sequence 1 MSQLEPRQQA FRNAMAHLSA AVNVITSNGP AGRCGITATA VCSVTDSPPT 50 51 LMLCINRNSE MNTVFKANGR LCVNVLSGEH EEVARHFAGM TEVPMERRFA 100 101 LHDWREGLAG LPVLHGALAN LQGRIAEVQE IGTHSVLLLE LEDIQVLEQG 150 151 DGLVYFSRSF HRLQCPRRAA 170

[0222] SEQ ID NO: 9: HpaC 1 atgtcccagc tcgaacccag gcagcaagcc ttccgcaacg ccatggcgca tctttcggcg 61 gcggtcaacg tgatcaccag caacggcccg gccggacgct gcgggatcac cgccaccgcg 121 gtctgctcgg tcaccgacag cccgccgacg ctgatgctct gcatcaaccg caacagcgag 181 atgaacacgg tgttcaaggc caacggtcga ctctgcgtga acgtcctcag cggcgaacat 241 gaagaggtgg cccgccactt cgccggcatg accgaggtcc cgatggaacg ccgcttcgcc 301 ctccacgact ggcgcgaggg cctcgccggg ttgccggtgc tgcacggcgc cctggccaac 361 ctgcagggac gcatcgccga ggtccaggag atcggcaccc actcggtgct gttgctggaa 421 ctggaggaca tccaggtcct cgaacagggc gacggcctgg tctacttcag ccgcagcttc 481 catcgcctgc aatgccccccg gcgggcggcc tga

[0223] SEQ ID NO: 10: HpaC primer (forward) forward :5′-ttccatatgtcccagctcgaacccaggcag-3′

[0224] SEQ ID NO: 11: HpaC primer (reverse) reverse : 5′-ttcggtacctcaggccgcccgccgggggca-3′ [Industrial Applicability]

[0225] According to the present invention, it is possible to efficiently synthesize β-Ala-DOPA by combining L-dopa with β-Ala. The β-Ala-DOPA synthesized according to the present invention is thought to have improved stability in the human body (decomposition in the body is suppressed). Therefore, it is expected that the present invention will enable the efficient synthesis of β-Ala-DOPA, the peptide itself or its metabolites of which may contribute to the improvement of brain function.

[0226] The present invention enables a stable supply of β-Ala-DOPA. β-Ala-DOPA synthesized by the present invention is expected to be used as a pharmaceutical product as a DOPA derivative that provides long-term effects even with small doses.

Claims

1. A method for producing β-Ala-DOPA, comprising: Due to L-amino acid α-ligase activity, (1) A step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates. A method for producing β-Ala-DOPA, comprising:

2. A method for producing β-Ala-DOPA, comprising: Using a microbial strain expressing L-amino acid α-ligase activity, (1) A step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates. The method for producing β-Ala-DOPA according to claim 1, comprising:

3. 3. The method for producing β-Ala-DOPA according to claim 2, wherein the microbial strain is a peptidase-deficient strain.

4. A method for producing β-Ala-DOPA, comprising: Using a protein with L-amino acid α-ligase activity, (1) A step of synthesizing β-Ala-DOPA from β-alanine (β-Ala) and L-dopa (L-DOPA) as substrates in the presence of adenosine triphosphate (ATP). The method for producing β-Ala-DOPA according to claim 1, comprising:

5. 5. The method for producing β-Ala-DOPA according to claim 1, which is carried out in the presence of an antioxidant.

6. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using L-amino acid α-ligase activity; and (2) A step of hydroxylating β-Ala-Tyr obtained in the step (1) by the activity of a two-component flavin-dependent monooxygenase to synthesize β-Ala-DOPA. A method for producing β-Ala-DOPA, comprising:

7. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates using a microbial strain expressing L-amino acid α-ligase activity; and (2) a step of synthesizing β-Ala-DOPA by hydroxylating β-Ala-Tyr obtained in the step (1) using a microbial strain expressing two-component flavin-dependent monooxygenase activity; The method for producing β-Ala-DOPA according to claim 6, comprising:

8. 8. The method for producing β-Ala-DOPA according to claim 7, wherein the microbial strain is a peptidase-deficient strain.

9. A method for producing β-Ala-DOPA, comprising: (1) synthesizing β-Ala-Tyr from β-alanine (β-Ala) and L-tyrosine (L-Tyr) as substrates in the presence of adenosine triphosphate (ATP) using a protein having L-amino acid α-ligase activity; and (2) using a protein having two-component flavin-dependent monooxygenase activity, in the presence of reduced nicotinamide adenine dinucleotide (NADH), to hydroxylate β-Ala-Tyr obtained in the step (1) to synthesize β-Ala-DOPA; The method for producing β-Ala-DOPA according to claim 6, comprising:

10. The method for producing β-Ala-DOPA according to any one of claims 6 to 9, which is carried out in the presence of an antioxidant.

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