L-glutamate oxidase, and method for producing α-ketoglutaric acid or hydroxypipecolic acid

Abstract

The present invention addresses the problem of providing a glutamate oxidase that efficiently converts glutamic acid to α-ketoglutaric acid. The present invention provides the following novel glutamate oxidase.　A glutamate oxidase having a polypeptide, the glutamate oxidase including any of the following amino acid sequences (1), (2), and (3): (1) An amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in an amino acid sequence represented by SEQ ID NO: 2 or 4; and (3) an amino acid sequence having 90% or greater sequence identity with an amino acid sequence represented by SEQ ID NO: 2 or 4.

Description

Method for producing L-glutamate oxidase and α-ketoglutaric acid or hydroxypipecolic acid

[0001] This invention relates to a novel L-glutamate oxidase. Furthermore, this invention relates to a method for producing α-ketoglutaric acid by oxidizing glutamate using this L-glutamate oxidase. Moreover, this invention relates to a method for producing hydroxypipecolic acid using the produced α-ketoglutaric acid.

[0002] In particular, this invention relates to an L-glutamate oxidase that can maintain its enzymatic activity even in the presence of α-ketoglutaric acid or ammonium ions, and to a technology for producing α-ketoglutaric acid or hydroxypipecolic acid on an industrial scale, at low cost and with high efficiency, using this L-glutamate oxidase.

[0003] α-Ketoglutaric acid (hereinafter sometimes referred to as "αKG") is known as an intermediate product of the citric acid cycle (TCA cycle) and plays an important role in the synthesis of amino acids and proteins, energy metabolism, etc. αKG is also useful as a raw material in pharmaceutical manufacturing; for example, it is known to be usable as a raw material for pharmaceuticals used for neuropathic pain (Patent Document 1). L-Glutamate oxidase is known to be usable for the analysis of umami components in food and L-glutamic acid, a neurotransmitter in the brain (Patent Document 2). Furthermore, hydroxypipecolic acid is known to be a useful compound as an intermediate for the synthesis of pharmaceuticals (Patent Document 6).

[0004] It is known that αKG can be produced by oxidizing L-glutamic acid (hereinafter sometimes referred to as "L-Glu") using L-glutamic acid oxidase (hereinafter sometimes referred to as "GOX").

[0005] GOX is an enzyme belonging to EC 1.4.3.11 and catalyzes the following reaction: L-glutamic acid + O ２ +H ２ O → α-ketoglutaric acid + H ２ O ２ +NH３

[0006] In other words, GOX is an enzyme that oxidizes L-Glu to produce αKG, hydrogen peroxide, and ammonia. The ammonia produced exists as ammonium ions dissolved in water, the reaction solvent.

[0007] In addition, L-amino acid oxidase is another known enzyme that catalyzes the same reaction using the same substrate and product. However, while L-amino acid oxidase can use amino acids other than L-Glu as substrates, GOX is different from L-amino acid oxidase in that it exhibits very high substrate specificity for L-Glu.

[0008] Generally, methods for producing αKG from L-Glu using GOX have relatively mild reaction conditions and can obtain high yields while minimizing environmental impact (Patent Documents 2-5). For example, Patent Document 2 discloses a method for producing αKG using L-Glu as a substrate with GOX derived from Streptomyces sp. X-119-6 strain.

[0009] However, these conventional methods using GOX had a problem: the products, αKG and ammonium ions, inhibited the function of the enzyme (GOX), reducing reaction efficiency. This problem was particularly pronounced under conditions where these products were present in high concentrations, making efficient production on an industrial scale difficult.

[0010] Furthermore, αKG is known to function as an important comaid in the hydroxylation reaction of amino acids such as pipecolic acid using hydroxylase.

[0011] Hydroxidases are enzymes that introduce oxygen into the C-H bond of a substrate to facilitate a hydroxylation reaction. A typical example of a hydroxylase is αKG-dependent dioxygenase. αKG-dependent dioxygenase is known to catalyze the hydroxylation reaction of pipecolic acid in the synthesis of hydroxypipecolic acid, for example, in the synthesis of pipecolic acid used as an intermediate for the synthesis of pharmaceuticals (Patent Document 6). Since αKG functions as an important co-matrix in the hydroxylation reaction of pipecolic acid, it is thought that the efficient supply of αKG to the reaction system improves the efficiency of the reaction.

[0012] Furthermore, in order to promote the reaction of αKG-dependent dioxygenase, FeSO ４ It is known that the addition of ferrous ions such as is important. The addition of ferrous ions improves the efficiency of the reaction. However, as described in Non-Patent Document 2, ferrous ions may inhibit other enzymatic reactions, particularly the GOX reaction. Therefore, it is considered difficult to carry out a sequential reaction in which αKG is produced by GOX, and then hydroxypipecolic acid is obtained using the resulting αKG with αKG-dependent dioxygenase.

[0013] Furthermore, Non-Patent Document 1 describes the production of cis-4-hydroxy-L-proline using αKG generated from L-Glu with GOX. In this technology, the accumulation concentration of αKG is low, so it is thought that the two steps of αKG generation and hydroxylation reaction must be carried out with different bacterial strains in order to efficiently supply αKG to the reaction system. This complicates the overall reaction process and is thought to lead to a decrease in the production efficiency of cis-4-hydroxy-L-proline. Non-Patent Document 2 also reports a method for synthesizing hydroxyamino acids by combining an oxidation reaction to generate αKG from L-Glu using GOX with the hydroxylation reaction of amino acids such as isoleucine. In this technology, although the accumulation concentration of αKG is high, it is thought that a continuous reaction is difficult because the oxidation reaction for αKG generation and the subsequent amino acid hydroxylation reaction must be carried out in two stages in order to efficiently carry out the oxidation reaction.

[0014] Therefore, based on these prior art documents, combining the oxidation reaction for αKG production with the hydroxylation reaction of amino acids presents challenges such as a complex overall reaction process and difficulty in achieving continuous reactions, making it difficult to scale up the reaction or apply it to industrial production.

[0015] International Publication No. 2014 / 155291 Brochure, International Publication No. 2001 / 079503 Brochure, Specification of Chinese Patent No. 107686850, Specification of Chinese Patent No. 106318888, Specification of Chinese Patent No. 104745545, International Publication No. 2017 / 057730 Brochure

[0016] Chen et al. Microb Cell Fact (2017) 16:210 Nie et al. Int. J. Mol. Sci. 2020,21,5347

[0017] The present invention aims to provide a method for efficiently producing αKG using GOX by reducing enzyme inhibition by the product αKG and ammonium ions, thereby suppressing the decrease in reaction efficiency. Furthermore, it aims to provide a production method that, when combining an oxidation reaction using GOX for αKG production with an amino acid hydroxylation reaction using hydroxylase, enables efficient production of αKG under high-concentration product environments, which was difficult with conventional techniques, and allows the hydroxylation reaction after the oxidation reaction to be carried out efficiently and continuously as a series of processes. In addition, it aims to provide a method for producing αKG and hydroxyamino acids that can be scaled up and applied to industrial production.

[0018] The inventors, through diligent research to solve the above problems, have discovered a novel GOX with a specific amino acid sequence that exhibits low enzyme inhibition by the product αKG and ammonium ions (hereinafter sometimes referred to as "product inhibition") in the production of αKG using GOX. Furthermore, they have discovered a method for efficiently producing αKG by suppressing the decrease in reaction efficiency using this novel GOX. Moreover, they have found a production method that enables efficient production of αKG even when combining the oxidation reaction using GOX for αKG production with the hydroxylation reaction of amino acids using hydroxylase, and that allows the hydroxylation reaction after the oxidation reaction to be carried out efficiently and continuously as a series of processes. Furthermore, they have discovered a method for producing αKG and hydroxyamino acids that can be scaled up and applied to industrial production. The present invention was achieved based on these findings.

[0019] In other words, the gist of the present invention is as follows: [1] A glutamate oxidase having a polypeptide comprising any of the following amino acid sequences: (1) the amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 2 or 4; (3) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4. [2] A nucleic acid encoding any of the following amino acid sequences: (1) the amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 2 or 4; (3) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4. [3] A nucleic acid according to [2], comprising any of the following base sequences: (4), (5), or (6): (4) a base sequence represented by SEQ ID NO: 1 or 3; (5) a base sequence having one or more base substitutions, deletions, and / or additions in the base sequence represented by SEQ ID NO: 1 or 3; (6) a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1 or 3. [4] A recombinant vector comprising the nucleic acid according to [2] or [3]. [5] A recombinant vector according to [4], further comprising a nucleic acid encoding a hydroxylase and / or a nucleic acid encoding catalase. [6] A transformant comprising the recombinant vector according to [4]. [7] A method for producing α-ketoglutarate, comprising contacting glutamate oxidase according to [1], a microorganism or cell capable of producing the glutamate oxidase, a processed product of the microorganism or cell, and / or a culture medium containing the glutamate oxidase obtained by culturing the microorganism or cell with glutamate to produce α-ketoglutarate. [8] The method for producing α-ketoglutaric acid according to [7], wherein the microorganism or cell is the transformant described in [6].A method for producing hydroxypipecolic acid, comprising hydroxylating pipecolic acid with a hydroxylase in the presence of α-ketoglutaric acid, wherein the α-ketoglutaric acid is obtained by the production method described in [7]. A method for producing hydroxypipecolic acid.

[10] The method for producing hydroxypipecolic acid according to [9], wherein the microorganism or cell is a transformant that co-expresses the hydroxylase and the glutamate oxidase and / or catalase.

[0020] According to the present invention, in the production of αKG using GOX, a GOX with low enzyme inhibition by the product αKG or ammonium ions can be provided. Further, by using the GOX of the present invention, it is possible to provide a method for efficiently producing αKG by suppressing a decrease in reaction efficiency. Furthermore, even when an oxidation reaction using GOX for αKG generation and a hydroxylation reaction of an amino acid using a hydroxylase are combined, efficient production of αKG is possible, and a production method capable of efficiently and continuously performing the hydroxylation reaction after the oxidation reaction as a series of processes can be provided. Furthermore, a method for producing αKG and hydroxyamino acids applicable to scale-up and industrial production can be provided.

[0021] In addition, the GOX of the present invention can also be suitably used for the analysis of umami components in foods and L-glutamic acid, which is a neurotransmitter in the brain.

[0022] It is a figure showing the relationship between the αKG concentration and the absorbance at a wavelength of 250 nm in Reference Example 7. It is a figure showing the relationship between the relative activity of GOX and the αKG concentration in Example 4. In Example 5, the figure showing the relationship between the relative activity of GOX and the NH ４ Cl concentration. It is a figure showing the relationship between the relative activity of GOX and the presence or absence of a hydroxylation reaction substrate solution in Example 6. It is a figure showing the relationship between the passage of reaction time and the αKG production concentration in Example 7. It is a figure showing the relationship between the passage of reaction time and the L-Glu concentration and conversion rate in Example 8. It is a figure showing the relationship between the passage of reaction time and the conversion rate in Example 9.

[0023] 1. L-Glutamic acid oxidase of the present invention The GOX of the present invention has one of the following amino acid sequences (1), (2), or (3). (1) The amino acid sequence represented by SEQ ID NO: 2 or 4; (2) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of (1) above; (3) An amino acid sequence having substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence of (1) above.

[0024] The GOX of the present invention has the ability to oxidize L-Glu to produce αKG.

[0025] GOX of the present invention having the amino acid sequence of (1) The GOX of the present invention has the amino acid sequence represented by SEQ ID NO: 2 or 4.

[0026] The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence derived from Streptomyces chattanoogensis. The amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence derived from Streptomyces lydicus. These amino acid sequences are known amino acid sequences registered in databases such as the DNA Data Bank of Japan (DDBJ), but the ability to oxidize L-Glu to produce αKG has been discovered for the first time by the present inventors.

[0027] GOX of the present invention having the amino acid sequence of (2) A person skilled in the art can presume that it has the same effect as the amino acid sequence of (1) above. Therefore, examples of the GOX of the present invention include those having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (1) above. That is, the amino acid sequence of (2) is an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (1) above and has the ability to oxidize L-Glu to produce αKG. From the viewpoint of effects, preferably, it has an amino acid sequence having 95% or more, more preferably 98% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of (1) above.

[0028] In this specification, sequence identity refers to the percentage of matching nucleotides or amino acids shared between two sequences when the two sequences are aligned in an optimal manner. That is, identity can be calculated as (number of matching positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) pp. 403-410. More specifically, the search and analysis of the identity of nucleotide or amino acid sequences can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, CrystalW). When using a program, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. The specific methods of these analysis methods are well known to those skilled in the art.

[0029] (3) The GOX of the present invention having the amino acid sequence of (1) A person skilled in the art can presume that it will produce the same effects as the amino acid sequence of (1). Therefore, the GOX of the present invention includes an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence of (1). That is, the amino acid sequence of (2) is an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence of (1), and has the ability to oxidize L-Glu to produce αKG.

[0030] "One to more" means any number that does not impair the performance of the GOX of the present invention, and is usually 1 to 100 pieces, preferably 1 to 50 pieces, more preferably 1 to 20 pieces, even more preferably 1 to 10 pieces, and particularly preferably 1 to 5 pieces, from the viewpoint of effectiveness.

[0031] Also, in the case of substitution, conservative substitution that substitutes one amino acid with another biologically and / or chemically similar amino acid is preferred. For example, conservative substitutions include substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one basic amino acid with another basic amino acid, etc.

[0032] Here, according to Non-Patent Document 3, GOX derived from Streptomyces sp. X-119-6 strain is synthesized as a precursor-type single-chain polypeptide, and by protease treatment, a peptide containing a dozen or so amino acids at the N-terminus is removed, the structure is stabilized, and the enzyme activity is reported to be improved. Specifically, it has been shown that by treatment with a metalloendopeptidase derived from Streptomyces griseus, the precursor-type GOX is converted into the active-type GOX, thereby improving the catalytic efficiency and thermal stability of the enzyme. Thus, the dozen or so amino acids present at the N-terminus of GOX may cause the enzyme activity to be expressed or improved when removed by protease cleavage. The GOX of the present invention exhibits L-glutamic acid oxidase activity even when the dozen or so amino acids at the N-terminus are present, but it has been confirmed that higher activity is shown when the dozen or so amino acids at the N-terminus are removed. Therefore, when using the GOX of the present invention, it is preferably used in a form in which the dozen or so amino acids at the N-terminus are removed.

[0033] The GOX of the present invention has the following physicochemical properties. Action: Catalyzes the following reaction. L-glutamic acid + O ２ + H ２ O → α-ketoglutaric acid + H ２ O ２ + NH ３ Substrate specificity: Specific to L-glutamic acid. Property: The enzyme activity is hardly inhibited even in the presence of a high concentration of αKG and / or in the presence of a high concentration of ammonium ions, and it has a high L-glutamic acid oxidase activity.

[0034] The GOX of the present invention exhibits high L-glutamate oxidase activity (relative activity with reference activity being L-glutamate oxidase activity under conditions of αKG concentration of 200 mmol / L) of 80% or more, preferably 90% or more, more preferably 100% or more, even more preferably 110% or more, and particularly preferably 120% or more, even in the presence of a high concentration of αKG of 800 mmol / L.

[0035] Furthermore, the GOX of the present invention exhibits high L-glutamate oxidase activity (relative activity with L-glutamate oxidase activity under conditions of ammonium ion concentration of 100 mmol / L as the reference activity) of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 100% or more, even in the presence of a high concentration of ammonium ions of 200 mmol / L.

[0036] For example, as shown in Example 4 below, the GOX of the present invention, derived from Streptomyces chattanoogenesis (hereinafter sometimes referred to as "ScGOX") and Streptomyces lydicus (hereinafter sometimes referred to as "SlGOX"), maintain higher relative activity than the comparative example, the conventionally known Streptomyces sp. X-119-6 derived GOX (hereinafter sometimes referred to as "SxGOX"), under conditions of an αKG concentration of 800 mmol / L. In particular, ScGOX shows a relative activity of 120%, indicating that the GOX of the present invention is less affected by enzyme activity inhibition by αKG.

[0037] Furthermore, as shown in Example 5 described later, the GOX of the invention, ScGOX and SlGOX, maintain higher relative activity than the conventionally known SxGOX, which is a comparative example, under conditions of ammonium ion concentration of 200 to 600 mmol / L. In particular, ScGOX shows a relative activity of 100% or more, indicating that the effect of enzyme activity inhibition by ammonium ions is small.

[0038] In this specification, L-glutamate oxidase activity (hereinafter sometimes referred to as "GOX activity") refers to the oxidation of L-Glu to αKG and hydrogen peroxide (H ２ O ２ ), and ammonia (NH ３ This refers to the catalytic action of enzymes that produce (

[0039] In the present invention, GOX activity (U / g) can be evaluated by the L-Glu reduction activity or αKG generation activity after adding L-Glu as a substrate to a reaction system containing GOX.

[0040] For example, at a suitable temperature (e.g., around 10°C to 45°C) and pressure (e.g., around atmospheric pressure), a reaction solution containing 10 mmol / L of L-Glu as a substrate is contacted (reacted) with a microorganism or cell capable of producing the target GOX, a processed product of said microorganism or cell, or a protein purified from said microorganism or cell, and the amount of L-Glu consumed or αKG produced is measured. This allows for the calculation of the L-Glu depletion activity (U / g) or αKG production activity (U / g) per unit of cell volume (unit: g or turbidity) or total protein volume (unit: g) added to the reaction solution. Here, the unit (U) represents the ability to consume 1 μmol of L-Glu or produce αKG per minute.

[0041] The L-Glu depletion activity can also be determined by contacting the target GOX with L-Glu in the reaction solution, measuring the L-Glu concentration, and calculating the difference (amount of decrease) from the L-Glu concentration in the reaction solution at the start of the reaction. The L-Glu concentration in the reaction solution can be determined by directly measuring the reaction solution using high-performance liquid chromatography (HPLC).

[0042] The αKG production activity can be determined by contacting the target GOX with L-Glu in the reaction solution and then measuring the amount and concentration of αKG produced from L-Glu. Alternatively, the amount and concentration of αKG produced in the reaction solution can be directly measured using HPLC.

[0043] Furthermore, αKG production activity can also be estimated and determined by hydrazone conversion of αKG in the reaction solution and measuring the absorbance of the resulting hydrazone compound, thereby predicting the amount and concentration of αKG produced in the reaction solution.

[0044] A specific method is as follows: A hydrazine compound such as semicarbazide hydrochloride (hereinafter sometimes referred to as "SCA") or 2,4-dinitrophenylhydrazine (hereinafter sometimes referred to as "DNPH") is added to a reaction solution containing αKG, reacts with αKG to produce a hydrazone compound, and measures the absorbance at a specific wavelength. By comparing the obtained absorbance with the absorbance of a control solution containing a hydrazone compound, the concentration and amount of hydrazone compound in the reaction solution, i.e., the concentration and amount of αKG in the reaction solution, can be estimated.

[0045] Specifically, for example, if a calibration curve is created in advance by confirming the relationship between the concentration or amount of αKG in the reaction solution and the absorbance, the concentration and amount of αKG produced in the reaction solution can be estimated from the measured absorbance using that calibration curve.

[0046] As controls, for example, an αKG-containing reaction solution in which the reaction has not proceeded, an αKG-containing reaction solution without the addition of enzymes, an αKG-containing reaction solution with inactivated hydroxylase added, an αKG-containing reaction solution with recombinant cell lysate that does not express hydroxylase added, or an αKG-containing reaction solution in which the reaction was stopped at a reaction time of 0 can be used.

[0047] The wavelength used to measure absorbance is preferably near the wavelength that yields the maximum extinction coefficient of the hydrazone compound produced, and can be appropriately selected depending on the hydrazine compound used. For example, when using SCA, a wavelength of around 250 nm is preferred, and when using DNPH, a wavelength of around 430 nm is preferred. It is also preferable to select a wavelength that is less affected by other components contained in the reaction solution.

[0048] The amount of hydrazine compound used is not particularly limited, as long as it can react sufficiently with αKG, but it is usually at least 1 molar amount of αKG, preferably 2 molar amounts to 10,000 molar amounts, and more preferably 5 molar amounts to 1,000 molar amounts.

[0049] The pH when reacting αKG with the hydrazine compound is not particularly limited, as long as it is the pH at which αKG and the hydrazine compound react, but it is usually in the range of 1 to 10, preferably in the range of 2 to 8, and more preferably in the range of 3 to 7.

[0050] The reaction temperature is not particularly limited, as long as it is the temperature at which αKG and the hydrazine compound react, but it is usually in the range of 0°C to 100°C, preferably in the range of 10°C to 80°C, and more preferably in the range of 20°C to 60°C.

[0051] The reaction time can be appropriately selected depending on the reaction temperature and is not particularly limited as long as it is the time required for αKG and the hydrazine compound to react. Longer times are not a problem, but it is usually 0 minutes (measured immediately after addition) to 24 hours, and to obtain stable results, it is preferably 10 minutes to 2 hours, more preferably 30 minutes to 1 hour.

[0052] Furthermore, the GOX of the present invention has the characteristic of exhibiting little enzyme inhibition (product inhibition) by the product αKG and ammonium ions in the production of αKG using GOX, and especially little product inhibition in the presence of high concentrations of αKG and / or high concentrations of ammonium ions.

[0053] Here, the product inhibition of GOX can be evaluated, for example, by gradually increasing the concentration of αKG or ammonium ions in the reaction solution and measuring the change in GOX activity at each concentration. If the GOX being measured maintains its activity even in the presence of high concentrations of the product (αKG or ammonium ions), the product inhibition of GOX can be evaluated as small. If the GOX does not maintain its activity in the presence of low concentrations of the product (αKG or ammonium ions), the product inhibition of GOX can be evaluated as large.

[0054] Furthermore, the evaluation of ammonium ion product inhibition is performed using NH ４A method can be used to supply ammonium ions derived from inorganic salts such as Cl (ammonium chloride) into the reaction solution. This method allows for the stable supply of ammonium ions into the reaction solution at a specific concentration, enabling the measurement of GOX activity at a specific ammonium ion concentration and accurate evaluation of product inhibition of GOX by ammonium ions.

[0055] The GOX of the present invention can be produced from the amino acid sequence represented by SEQ ID NO: 2 or 4 by methods known to those skilled in the art, such as site-directed mutagenesis, PCR, and other well-known techniques.

[0056] Furthermore, the GOX of the present invention can also be produced by culturing a transformant containing the nucleic acid encoding it, and then separating and purifying the GOX from the resulting culture. The nucleic acid encoding the amino acid sequence of the GOX of the present invention may be DNA, RNA, or a DNA / RNA chimera. DNA is preferred. The nucleic acid may also be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of single-stranded, it may be a sense strand (i.e., coding strand) or an antisense strand (i.e., non-coding strand).

[0057] Examples of DNA that can be used as the basis for the DNA encoding the amino acid sequence of GOX in the present invention include DNA cloned from Streptomyces chattanoogenesis or Streptomyces lydicus. For example, it can be obtained from a DNA fraction prepared from cells or tissues derived from Streptomyces chattanoogenesis or Streptomyces lydicus by known PCR or hybridization methods. Alternatively, examples include full-length GOX cDNA directly amplified by Reverse Scriptase-PCR using a total RNA or mRNA fraction prepared from cells or tissues derived from Streptomyces chattanoogenesis or Streptomyces lydicus as a template. Such DNA can be transformed (mutated) using known methods such as ODA-LA PCR, Gapped duplex, Kunkel, or similar methods, using known kits, such as Mutan®-Super Express Km (TAKARA BIO INC.), Mutan® K (TAKARA BIO INC.), etc., to obtain the DNA encoding GOX of the present invention. Alternatively, the cDNA can be obtained by cloning cDNA from a cDNA library prepared by inserting the above-mentioned total RNA or mRNA fragments into a suitable vector, using colony or plaque hybridization or PCR, etc., and then transforming the cloned cDNA according to the above-mentioned method. The vector used in the library may be a bacteriophage, plasmid, cosmid, phagemid, etc.

[0058] Nucleic acids encoding the amino acid sequence of GOX according to the present invention include, for example, those comprising the following base sequences: (4) the base sequence represented by SEQ ID NO: 1 or 3; (5) a base sequence having one or more base substitutions, deletions, and / or additions in the base sequence represented by SEQ ID NO: 1 or 3; (6) a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1 or 3.

[0059] In the base sequence represented by (4) above, the base sequence represented by Sequence ID No. 1 is a synthetic base sequence in which the codons of the gene of Streptomyces chattanoogenesis are optimized for expression in E. coli, and the base sequence represented by Sequence ID No. 3 is a synthetic base sequence in which the codons of the gene of Streptomyces lydicus are optimized for expression in E. coli. Furthermore, not only the genes of Streptomyces chattanoogenesis or Streptomyces lydicus, but also nucleic acids in which the codons have been optimized according to the host being transformed, are naturally included in the nucleic acids encoding the GOX activity protein of the present invention.

[0060] The base sequence represented in (5) above includes a base sequence having one or more base substitutions, deletions, and / or additions in the base sequence represented in (4) above. In the case of substitutions, a conservative substitution is preferred, in which one base is substituted with another base that is biologically and / or chemically similar.

[0061] Here, "one to more" means any number such that the performance of the nucleic acid encoding the amino acid sequence of GOX in the present invention is not impaired, and is usually 1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5, from the viewpoint of effectiveness.

[0062] The base sequence represented in (5) is a base sequence having 90% or more sequence identity with the base sequence represented in (4), and from the viewpoint of effectiveness, a base sequence having 95% or more, more preferably 98% or more, and particularly preferably 99% or more sequence identity is mentioned.

[0063] Furthermore, as nucleic acids encoding the amino acid sequence of GOX according to the present invention, those including a base sequence that hybridizes with the complementary strand of the base sequence represented in (4) above under stringent conditions are also included in the nucleic acids of the present invention, insofar as they encode a polypeptide having GOX activity.

[0064] Here, "base sequences that hybridize under stringent conditions" refers to DNA base sequences obtained by using DNA as a probe and employing methods such as colony hybridization, plaque hybridization, or Southern blot hybridization under stringent conditions.

[0065] Stringent conditions include, for example, in colony hybridization or plaque hybridization, using a filter immobilized with DNA or fragments of DNA derived from colonies or plaques, hybridization being performed at 65°C in the presence of a 0.7 mol / L to 1 mol / L aqueous sodium chloride solution, followed by washing the filter at 65°C with a 0.1×SSC solution (composed of 1×SSC being 150 mmol / L aqueous sodium chloride solution and 15 mmol / L aqueous sodium citrate solution). This hybridization can be carried out according to the method described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989., etc.

[0066] Furthermore, the nucleic acids encoding the amino acid sequence of GOX in the present invention also include various nucleic acids derived from codon degeneracy, as long as they encode polypeptides that possess GOX activity. Codon degeneracy refers to the phenomenon in the translation process from nucleic acids to amino acids where different codons (base sequences consisting of three nucleotides) encode the same amino acid. For example, for alanine (A), the four different codons GCT, GCC, GCA, and GCG encode the same alanine. By utilizing this codon degeneracy, even if the amino acid sequence of a polypeptide is the same, the nucleic acid sequence encoding it can have diversity within the range of codon degeneracy, resulting in the production of the same polypeptide. Nucleic acid sequences encoding a particular identical polypeptide are equivalent to each other. Methods for obtaining these equivalent nucleic acid sequences include site-directed mutagenesis, as described later, modification of nucleic acid sequences using known genetic engineering techniques, or the use of artificially synthesized nucleic acids.

[0067] For example, a person skilled in the art can obtain a nucleic acid encoding the amino acid sequence of GOX of the present invention by appropriately performing substitutions, deletions, insertions and / or additions to the nucleic acid represented by Sequence ID No. 1 or 3 using site-directed mutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning, PCR A Practical Approach IRL Press pp. 200 (1991)), etc., to introduce base substitutions that produce the desired mutation.

[0068] Examples of the amino acid sequence of GOX and the nucleic acid encoding the amino acid sequence of GOX according to the present invention include, but are not limited to, the sequences listed in Table 1 below, which are also described in the examples below.

[0069]

[0070] In the method for producing αKG of the present invention, as described later, GOX may be used directly in the reaction with L-Glu as the substrate, but it is preferable to use a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and / or a culture medium containing the enzyme obtained by culturing the microorganism or cell.

[0071] The microorganism or cell capable of producing GOX in the present invention may be a microorganism or cell that originally possesses the ability to produce GOX, or a microorganism or cell that has been given the ability to produce GOX through breeding. The microorganism or cell may be alive or dead, and for example, dormant cells can be suitably used. Examples of the types of microorganisms or cells capable of producing GOX in the present invention include those described later as "host microorganisms" or "host cells."

[0072] As means of conferring the aforementioned production capacity through breeding, known methods such as genetic modification (transformation) and mutation treatment can be employed. Methods of transformation include introducing the target DNA and modifying expression regulatory sequences such as promoters on the chromosome to enhance the expression of the target DNA.

[0073] Of these, it is preferable to use a nucleic acid encoding the amino acid sequence of the present invention, and in particular, a microorganism or cell transformed with DNA encoding the amino acid sequence of the present invention.

[0074] As described above, the DNA encoding GOX in the present invention can be obtained by cloning and converting it using PCR with appropriate primers, with chromosomal DNA derived from Streptomyces chattanoogensis or Streptomyces Micromonospora chokoriensis as a template.

[0075] Furthermore, the DNA encoding GOX of the present invention can be obtained by preparing full-length GOX cDNA by directly amplifying total RNA or mRNA derived from Streptomyces chattanoogenesis or Streptomyces Micromonospora chokoriensis using RT-PCR as a template, and then cloning and converting it by performing PCR using appropriate primers.

[0076] For example, a recombinant vector of the present invention is provided by inserting the DNA encoding the GOX of the present invention, obtained as described above, into a known expression vector in an expressible configuration. Then, by transforming host cells with the recombinant vector, a transformant can be obtained into which the DNA encoding the protein of the present invention has been introduced. The transformant can also be obtained by incorporating the DNA encoding the protein of the present invention into the host's chromosomal DNA in an expressible manner using methods such as homologous recombination.

[0077] In this specification, "expression vector" refers to a genetic element used to replicate and express a protein having a desired function in a host organism by incorporating a polynucleotide encoding the protein having a desired function and introducing it into the host organism. Examples include, but are not limited to, plasmids, viruses, phages, and cosmids. Preferably, the expression vector is a plasmid.

[0078] In this specification, "transformed organism" means a microorganism or cell into which a target gene has been introduced using the expression vector, etc., and which has become capable of expressing a desired trait related to a protein having a desired function.

[0079] Examples of methods for producing transformants include introducing the DNA encoding GOX of the present invention into a plasmid vector, phage vector, or viral vector that is stably present in a host cell, and then introducing the constructed recombinant vector into the host cell, or directly introducing the DNA into the host genome and transcribing and translating its genetic information. In this case, it is preferable to ligate a suitable promoter upstream of the 5'- side of the DNA in the host, and it is even more preferable to ligate a terminator downstream of the 3'- side. Such promoters and terminators are not particularly limited as long as they are promoters and terminators known to function in the cells used as the host, and for example, vectors, promoters, and terminators detailed in "Basic Microbiology Course 8: Genetic Engineering" (Kyoritsu Shuppan) can be used.

[0080] The host microorganisms to be transformed to express GOX of the present invention are not particularly limited as long as the host itself does not adversely affect the substrate L-Glu or the target product αKG. For example, the following microorganisms can be cited.

[0081] Bacteria belonging to genera such as Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, and Lactobacillus have established host-vector systems.

[0082] Actinomycetes belonging to genera such as Rhodococcus and Streptomyces, for which host-vector systems have been established.

[0083] Yeast species belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, and Candida, for which host-vector systems have been established.

[0084] These are fungi belonging to genera such as Neurospora, Aspergillus, Cephalosporium, and Trichoderma, for which host-vector systems have been established.

[0085] The procedures for producing transformants, constructing recombinant vectors adapted to the host, and culturing the host can be carried out in accordance with techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, the method described in Green et al., Molecular Cloning: A Laboratory Manual (4th ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)).

[0086] The following are specific examples of preferred host microorganisms, preferred transformation methods for each microorganism, vectors, promoters, terminators, etc., but the present invention is not limited to these examples.

[0087] In the genus Escherichia, particularly Escherichia coli, plasmid vectors include pBR and pUC plasmids, as well as promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (fusion of lac and trp), λ phage PL, PR, and T7 phage. Terminators include those derived from trpA, phage, and rrnB ribosomal RNA.

[0088] In the Bacillus genus, suitable vectors include pUB110 plasmids and pC194 plasmids, and they can also be integrated into chromosomes. Promoters and terminators for enzyme genes such as alkaline proteases, neutral proteases, and α-amylases can be used.

[0089] In the genus Pseudomonas, examples of vectors include common host vector systems established for Pseudomonas putida and Pseudomonas cepacia, as well as plasmids involved in the degradation of toluene compounds, and broad-host-range vectors based on TOL plasmids (containing genes necessary for autonomous replication derived from RSF1010, etc.) such as pKT240 (Gene, 26, 273-82 (1983)).

[0090] For the genus Brevibacterium, particularly Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene 39, 281 (1985)) can be used. Various promoters and terminators used in E. coli can be utilized as promoters and terminators.

[0091] For the genus Corynebacterium, particularly Corynebacterium glutamicum, suitable vectors include plasmid vectors such as pCS11 (Japanese Patent Publication No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)).

[0092] For the genus Saccharomyces, particularly Saccharomyces cerevisiae, suitable vectors include YRp, YEp, YCp, and YIp plasmids. Additionally, promoters and terminators for various enzyme genes, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase, are available.

[0093] In the genus Schizosaccharomyces, suitable vectors include plasmid vectors derived from Schizosaccharomyces pombe, as described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Bio Inc. and is readily available.

[0094] Within the genus Aspergillus, Aspergillus niger and Aspergillus oryzae are among the most well-studied fungi, and plasmid and chromosomal integration are available, as are promoters derived from extracellular proteases and amylases (Trendsin Biotechnology 7, 283-287 (1989)).

[0095] In addition, host vector systems tailored to various microorganisms have been established and can be used as appropriate.

[0096] Furthermore, in addition to microorganisms, various host-vector systems have been established in plants and animals. In particular, systems for expressing large quantities of heterologous proteins in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)), in plants such as rapeseed, corn, and potatoes, and systems using cell-free protein synthesis systems such as E. coli cell-free extracts and wheat germ have been established and are suitable for use.

[0097] Examples of processed products of microorganisms or cells capable of producing GOX according to the present invention include cell preparations obtained by treating the microorganisms or cells with organic solvents or surfactants such as acetone, dimethyl sulfoxide, and toluene, freeze-drying, or physically or enzymatically disrupting them; purified enzymes (including partially purified enzymes) obtained by extracting enzyme fractions from microorganisms or cells as crude or purified products; and products in which these are immobilized on carriers such as polyacrylamide gel and carrageenan gel.

[0098] Examples of culture media containing the enzyme obtained by culturing microorganisms or cells capable of producing the GOX of the present invention include a suspension of the microorganisms or cells with a liquid medium, or, if the microorganisms or cells are secretory expression cells, the supernatant or concentrate thereof obtained by removing the microorganisms or cells by centrifugation or the like. The culture conditions can be any conditions suitable for culturing microorganisms or cells, and may also be conditions that have been appropriately adjusted to optimize the activity, physical properties, production capacity, etc., of the GOX of the present invention. For example, when using Escherichia coli as a host, it is preferable to culture at a culture temperature of 15°C to 37°C for about 12 to 48 hours.

[0099] In the preparation of DNA fragments in the present invention, that is, obtaining DNA fragments by gene synthesis, preparing recombinant vectors using the obtained DNA fragments, and producing GOX using the recombinant vectors, other known techniques may also be used.

[0100] 2. Recombinant Vectors and Transformants of the Present Invention The recombinant vector of the present invention comprises a nucleic acid encoding an amino acid sequence possessed by GOX of the present invention, i.e., any of the following amino acid sequences: (1) an amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 2 or 4; (3) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4.

[0101] The recombinant vector of the present invention is preferably a nucleic acid that encodes the amino acid sequence of GOX of the present invention and includes a nucleic acid containing any of the following base sequences: (4) the base sequence represented by SEQ ID NO: 1 or 3; (5) a base sequence having one or more base substitutions, deletions, and / or additions in the base sequence represented by SEQ ID NO: 1 or 3; (6) a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1 or 3.

[0102] The recombinant vector of the present invention may further contain, in addition to the nucleic acid encoding the amino acid sequence of the GOX of the present invention, a nucleic acid encoding catalase and / or a nucleic acid encoding hydroxylase.

[0103] Here, catalase is H ２ O ２ An enzyme having decomposition activity. For example, catalase derived from Escherichia coli K-12 MG1655 can be cited. Examples of catalases include polypeptides having catalase activity and comprising any of the following amino acid sequences: (7) the amino acid sequence represented by Sequence ID No. 8; (8) an amino acid sequence having one to more (1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5) amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by Sequence ID No. 8; (9) an amino acid sequence having 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more) sequence identity with the amino acid sequence represented by Sequence ID No. 8.

[0104] Furthermore, hydroxylases are enzymes that introduce oxygen into the C-H bond of a substrate to carry out a hydroxylation reaction. A typical example of a hydroxylase is αKG-dependent dioxygenase. In the present invention, enzymes having the activity to hydroxylate pipecolic acid to produce hydroxypipecolic acid are preferred. Examples include pipecolic acid hydroxylase derived from Micromonospora chokoriensis, pipecolic acid hydroxylase derived from Xenorhabdus doucetiae, and pipecolic acid hydroxylase derived from Fusarium oxysporum, which hydroxylates L-pipecolic acid to convert it to (5S)-hydroxy-L-pipecolic acid. Examples of hydroxylases include polypeptides having hydroxylase activity and containing any of the following amino acid sequences: (10), (11), or (12). (10) an amino acid sequence represented by SEQ ID NO: 10 or 12; (11) an amino acid sequence having one to more (1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5) amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 10 or 12; (12) an amino acid sequence having 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more) sequence identity with the amino acid sequence represented by SEQ ID NO: 10 or 12.

[0105] The recombinant vector of the present invention may contain a nucleic acid encoding the amino acid sequence of the GOX of the present invention. Alternatively, the recombinant vector of the present invention may contain a nucleic acid encoding the amino acid sequence of the GOX of the present invention and a nucleic acid encoding catalase, or a nucleic acid encoding the amino acid sequence of the GOX of the present invention and a nucleic acid encoding hydroxylase. Furthermore, the recombinant vector of the present invention may contain a nucleic acid encoding the amino acid sequence of the GOX of the present invention, a nucleic acid encoding catalase, and a nucleic acid encoding hydroxylase.

[0106] The transformant of the present invention contains the recombinant vector described above.

[0107] The transformants of the present invention can be obtained by transforming host cells or host microorganisms with a recombinant vector containing a nucleic acid encoding the amino acid sequence of the present invention GOX, a recombinant vector containing a nucleic acid encoding the amino acid sequence of the present invention GOX and a nucleic acid encoding catalase, a recombinant vector containing a nucleic acid encoding the amino acid sequence of the present invention GOX and a nucleic acid encoding hydroxylase, or a recombinant vector containing a nucleic acid encoding the amino acid sequence of the present invention GOX, a nucleic acid encoding catalase and a nucleic acid encoding hydroxylase.

[0108] In the method for producing αKG of the present invention, described later, transformants obtained by transforming host cells or host microorganisms with a recombinant vector containing a nucleic acid encoding the amino acid sequence of GOX of the present invention can be used.

[0109] Furthermore, in the method for producing αKG of the present invention, transformants obtained by transforming host cells or host microorganisms with a recombinant vector containing nucleic acids encoding the amino acid sequence of GOX of the present invention and nucleic acids encoding catalase can be used, that is, transformants in which GOX and catalase of the present invention are co-expressed within the same host cell or host microorganism. By using such transformants, H ２ O ２ Because the degradation of αKG by [unspecified factor] can be suppressed, αKG can be produced in high yield.

[0110] Furthermore, in the present invention, when the production of αKG and hydroxypipecolic acid are carried out consecutively or in the same reaction solution, a transformant obtained by transforming a host cell or host microorganism with a recombinant vector containing nucleic acid encoding the amino acid sequence of GOX of the present invention, nucleic acid encoding catalase, and nucleic acid encoding hydroxylase, that is, a transformant in which GOX, catalase, and hydroxylase of the present invention are co-expressed in the same host cell or host microorganism, can be used. In this case, the reaction can be carried out by adding L-Glu and pipecolic acid to the reaction system. That is, αKG converted from L-Glu by GOX is used as a cosubstrate for the hydroxylation reaction of pipecolic acid to hydroxypipecolic acid by hydroxylase, so the reaction is carried out efficiently. By using this transformant, hydroxypipecolic acid can be produced industrially at low cost and inexpensively.

[0111] 3. Method for producing α-ketoglutaric acid of the present invention The method for producing αKG of the present invention involves contacting L-Glu with GOX of the present invention, microorganisms or cells capable of producing GOX of the present invention, processed products of said microorganisms or cells, and / or a culture medium containing GOX of the present invention obtained by culturing said microorganisms or cells (hereinafter sometimes referred to as "GOX of the present invention, etc.") to produce αKG.

[0112] In the production method of the present invention, purified or crudely purified GOX of the present invention, microorganisms or cells capable of producing GOX of the present invention (for example, transformants having DNA encoding GOX of the present invention), processed products of said microorganisms or cells, and / or a culture medium containing GOX of the present invention obtained by culturing said microorganisms or cells can be used. Of these, it is preferable to use microorganisms or cells capable of producing GOX of the present invention, processed products of said microorganisms or cells, and / or a culture medium containing GOX of the present invention obtained by culturing said microorganisms or cells (for example, transformants having DNA encoding GOX of the present invention), and it is more preferable to use transformants having DNA encoding GOX of the present invention. In the production method of the present invention, multiple types of GOX may be used in combination.

[0113] The amount of GOX, etc., of the present invention to be brought into contact with L-Glu is not particularly limited, as long as it is an amount that can produce αKG. For example, when adding the microorganism or cells to the reaction solution containing L-Glu, the concentration of the microorganism or cells in the reaction solution is usually about 0.01 w / v% to 20 w / v%, preferably 1 w / v% to 10 w / v%, by wet bacterial weight. Here, w / v% means weight / volume%.

[0114] The L-Glu used in this invention is not limited to the free amino acid form (free acid form), but other salts, derivatives, etc. can be used depending on the reaction conditions and purpose. For example, the free acid form of L-Glu, the sodium salt, potassium salt, or calcium salt of L-Glu, and L-Glu bonded to a chlorine compound can be used. Those skilled in the art can select the optimal form of L-Glu according to the reaction conditions and purpose, and implement the present invention without impairing its effects.

[0115] Alternatively, instead of L-Glu, an inexpensive compound that the host can metabolize, such as glucose, can be used, and the L-Glu produced in this process can be used in the reaction.

[0116] In the present invention, the amount of L-Glu used can be appropriately selected according to the amount of αKG to be produced. Furthermore, L-Glu can be used so that the substrate concentration in the reaction solution containing L-Glu and GOX of the present invention is typically 0.01 mol / L to 1.6 mol / L, preferably 0.2 mol / L to 0.8 mol / L, and more preferably 0.2 mol / L to 0.6 mol / L.

[0117] Furthermore, the method of contact is not particularly limited. For example, L-Glu and the GOX of the present invention may be added all at once at the beginning, or L-Glu, which will be the substrate, may be added continuously or intermittently to a liquid containing the GOX of the present invention. Alternatively, the GOX of the present invention may be added continuously or intermittently to a liquid containing the L-Glu, which will be the substrate. Upon contact between L-Glu and the GOX of the present invention, L-Glu is deaminated to produce αKG.

[0118] The aforementioned contact can usually be carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent, but it is preferable to carry it out in an aqueous medium from the viewpoint of reducing post-treatment and industrial burden.

[0119] Examples of aqueous media include water or known buffers such as Good's buffer, phosphate buffer, Tris buffer, or borate buffer. The pH of these buffers can be appropriately adjusted according to the reaction conditions. As for organic solvents, it is preferable to use those that have high solubility for the substrate L-Glu, such as methanol, ethanol, acetone, and dimethyl sulfoxide. Alternatively, organic solvents that can efficiently remove reaction byproducts may be used, such as ethyl acetate and butyl acetate.

[0120] The contact can be carried out under a pressure of, for example, atmospheric pressure, but pressurization can be applied if necessary. Furthermore, since this reaction requires oxygen, ventilation is preferable.

[0121] The temperature at contact is not particularly limited as long as it is the temperature at which L-Glu is oxidized and αKG is produced, but is usually 4°C to 60°C, preferably 10°C to 45°C, and particularly preferably 15°C to 30°C. Furthermore, contact can usually be carried out under conditions of pH 3 to 11, preferably pH 5 to 8. The contact time is not particularly limited as long as it is the time it takes for L-Glu to be oxidized and αKG to be produced, but is usually 10 minutes or more, preferably 30 minutes or more, and usually within 90 hours, preferably within 72 hours.

[0122] Here, as mentioned above, GOX is an enzyme that oxidizes L-Glu to produce αKG, hydrogen peroxide, and ammonia, but hydrogen peroxide (H ２ O ２ It is known that ) causes the degradation of αKG.

[0123] Therefore, in the present invention, catalase is used for H ２ O ２ It is preferable to suppress the decomposition of αKG by decomposing it and maintain the reaction efficiency. In this specification, catalase refers to hydrogen peroxide (H ２O ２ ) water (H ２ O) and oxygen (O) ２ It is an enzyme that breaks down (

[0124] The catalase used is H ２ O ２ As long as it has degradation activity, it is not particularly limited, and for example, catalase derived from Escherichia coli K-12 MG1655 can be used.

[0125] Methods for supplying the catalase include using endogenous catalase such as a transformant having DNA encoding GOX according to the present invention, adding purified or crudely purified catalase to the reaction system, and incorporating the nucleic acid encoding catalase into an expression vector and expressing it in E. coli. From a cost viewpoint, the method of incorporating the nucleic acid encoding catalase into an expression vector and expressing it in E. coli is preferred, and a method of simultaneously incorporating GOX and the nucleic acid encoding catalase into an expression vector and co-expressing them in E. coli is even more desirable.

[0126] By using catalase, H ２ O ２ This allows for efficient decomposition and generation of αKG. ２ O ２ This makes it possible to suppress the degradation of αKG, thereby enabling the production of αKG in high yield.

[0127] The generated αKG can be separated from the reaction solution by removing bacterial cells, proteins, etc., from the reaction solution using separation methods known to those skilled in the art, such as centrifugation or membrane treatment. Furthermore, the separated αKG can be purified by appropriately combining methods such as recrystallization under acidic or neutral conditions, recrystallization in water or ethanol solvent, distillation, column chromatography using ion exchange resin, reverse-phase chromatography, or crystallization at the isoelectric point.

[0128] Furthermore, the generated αKG can be used in reactions and production processes that utilize αKG, such as the production of hydroxypipecolic acid, without separation or purification from the reaction solution containing the generated αKG.

[0129] The present invention's method for producing αKG makes it possible to industrially produce αKG, which is useful as a pharmaceutical raw material, with high productivity, low cost, and low price.

[0130] 4. Method for Producing Hydroxypipecolic Acid of the Present Invention The method for producing hydroxypipecolic acid of the present invention is a method for producing hydroxypipecolic acid by contacting pipecolic acid with a hydroxylase, a microorganism or cell capable of producing the hydroxylase, a processed product of the microorganism or cell, and / or a culture medium containing the hydroxylase obtained by culturing the microorganism or cell (hereinafter collectively referred to as "hydroxylase, etc.") in the presence of αKG, characterized in that αKG is used as αKG obtained by the method for producing αKG described above.

[0131] In the method for producing hydroxypipecolic acid of the present invention, the αKG used may be any αKG obtained by the αKG production method described above. This can include a reaction solution containing αKG obtained by contacting L-Glu with GOX or the like of the present invention, a reaction solution containing crudely purified αKG from the reaction solution, αKG separated from the reaction solution, or purified αKG. However, since separating or purifying αKG is costly and time-consuming, it is preferable not to separate or purify αKG for industrial production. For industrial production, it is preferable to use a reaction solution containing αKG obtained by contacting L-Glu with GOX or the like of the present invention.

[0132] The amount of αKG used is usually equimolar to or greater than that of the substrate pipecolic acid. From the viewpoint of manufacturing cost, the amount of αKG used is preferably 0.8 times the molar amount of pipecolic acid or more, more preferably 0.9 times the molar amount or more, even more preferably 0.95 times the molar amount or more, and also preferably 5 times the molar amount or less, more preferably 3 times the molar amount or less, and even more preferably 2 times the molar amount or less. In the present invention, it is particularly preferable to use an amount equivalent to ±0.2 times the molar amount of the substrate pipecolic acid.

[0133] αKG may be added to the reaction system containing pipecolic acid and hydroxylase all at once at the beginning, or it may be added to the reaction system continuously or intermittently from the viewpoint of reducing the effect of inhibition on hydroxylase and improving the accumulation concentration of the product.

[0134] Examples of pipecolic acid include 3-pipecolic acid, 4-pipecolic acid, or 5-pipecolic acid. In the present invention, 5-pipecolic acid is preferred from the viewpoint of effectiveness.

[0135] Purified pipecolic acid may be used, or a solution containing pipecolic acid obtained by a bacterial reaction may be used.

[0136] Examples of hydroxypipecolic acid include 3-hydroxypipecolic acid, 4-hydroxypipecolic acid, or 5-hydroxypipecolic acid, and more specifically, (3S)-hydroxy-L-pipecolic acid, (3R)-hydroxy-L-pipecolic acid, (4S)-hydroxy-L-pipecolic acid, (4R)-hydroxy-L-pipecolic acid, (5S)-hydroxy-L-pipecolic acid, or (5R)-hydroxy-L-pipecolic acid. From the viewpoint of effectiveness, (5S)-hydroxy-L-pipecolic acid or (5R)-hydroxy-L-pipecolic acid is preferred, and (5S)-hydroxy-L-pipecolic acid is particularly preferred.

[0137] Hydroxylases are not particularly limited, as they are enzymes that introduce oxygen into the C-H bond of a substrate to carry out a hydroxylation reaction, but examples include αKG-dependent dioxygenase. In the present invention, an enzyme having the activity to hydroxylate pipecolic acid to produce hydroxypipecolic acid is preferred. For example, pipecolic acid hydroxylase derived from Micromonospora chokoriensis or Xenorhabdus doucetiae that hydroxylates L-pipecolic acid to (5S)-hydroxy-L-pipecolic acid can be used. Pipecolic acid hydroxylase derived from Fusarium oxysporum that hydroxylates L-pipecolic acid to (4S)-hydroxy-L-pipecolic acid can be used. Pipecolic acid hydroxylase derived from Segniliparus rugosus that hydroxylates L-pipecolic acid to (3S)-hydroxy-L-pipecolic acid can be used. In the method for producing hydroxypipecolic acid of the present invention, multiple types of hydroxylases may be used in combination.

[0138] In the method for producing hydroxypipecolic acid of the present invention, purified or crudely purified hydroxylase, microorganisms or cells capable of producing the hydroxylase (for example, transformants having DNA encoding the hydroxylase), processed products of the microorganisms or cells, and / or a culture medium containing the hydroxylase obtained by culturing the microorganisms or cells can be used. Of these, it is preferable to use microorganisms or cells capable of producing the hydroxylase, processed products of the microorganisms or cells, and / or a culture medium containing the hydroxylase obtained by culturing the microorganisms or cells. Industrially, it is more preferable to use transformants having DNA encoding the hydroxylase.

[0139] The amount of hydroxylase or the like that brought into contact with pipecolic acid is not particularly limited, as long as it is an amount that can hydroxylate pipecolic acid to produce hydroxypipecolic acid. For example, when adding the microorganism or cells to a reaction solution containing L-pipecolic acid, the amount added should be such that the concentration of the microorganism or cells in the reaction solution is usually about 0.1 w / v% to 50 w / v%, preferably 1 w / v% to 20 w / v%, by wet cell weight. When adding the treated product or culture medium to a reaction solution containing L-pipecolic acid, the specific activity of the hydroxylase to be used should be determined, and an amount should be added such that the cell concentration in the reaction solution becomes the cell concentration. Here, w / v% means weight / volume%.

[0140] The method of contact is not particularly limited; for example, L-pipecolic acid, which is the substrate, can be added to a liquid containing a hydroxylase or the like. Alternatively, a hydroxylase or the like may be added to a liquid containing pipecolic acid, which is the substrate. When pipecolic acid comes into contact with a hydroxylase or the like, pipecolic acid is hydroxylated to produce hydroxypipecolic acid.

[0141] The amount of pipecolic acid can be appropriately selected depending on the amount of hydroxypipecolic acid to be produced. Furthermore, pipecolic acid can be used as the substrate concentration in the reaction solution containing pipecolic acid and hydroxylase, etc., usually at 0.01 w / v% to 90 w / v%, preferably 0.1 w / v% to 30 w / v%.

[0142] Pipecolic acid and hydroxylases may be added all at once at the beginning, or they may be added continuously or intermittently to reduce the effects of enzyme substrate inhibition and to improve the accumulation concentration of the product.

[0143] The aforementioned contact can usually be carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent, but it is preferable to carry it out in an aqueous medium from the viewpoint of reducing post-treatment and industrial burden.

[0144] Examples of aqueous media include water or known buffers such as Good's buffer, phosphate buffer, Tris buffer, or borate buffer. The pH of these buffers can be appropriately adjusted according to the reaction conditions. As for organic solvents, it is preferable to use those that have high solubility for the substrate L-Glu, such as methanol, ethanol, acetone, and dimethyl sulfoxide. Alternatively, organic solvents that can efficiently remove reaction byproducts may be used, such as ethyl acetate and butyl acetate.

[0145] The contact can be carried out under a pressure of, for example, atmospheric pressure, but pressurization is also possible if necessary. Furthermore, since this reaction requires oxygen, ventilation is preferable. The temperature is not particularly limited as long as it is the temperature at which L-Glu is oxidized to produce αKG and pipecolic acid is hydroxylated to produce hydroxypipecolic acid, but it is usually in the range of 4°C to 60°C, preferably 10°C to 45°C, and particularly preferably 15°C to 30°C. In addition, the contact can usually be carried out under conditions of pH 3 to 11, preferably pH 5 to 8. The contact time is not particularly limited as long as it is the time required for L-Glu to be oxidized to produce αKG and for pipecolic acid to be hydroxylated to produce hydroxypipecolic acid, but it is usually 10 minutes or more, preferably 30 minutes or more, and usually within 90 hours, preferably within 72 hours.

[0146] Furthermore, the contact is preferably carried out in the presence of divalent iron ions. The divalent iron ions are usually used in the reaction solution at a concentration of 0.00001 mol / L to 0.1 mol / L, preferably in the range of 0.0001 mol / L to 0.01 mol / L. The divalent iron ions can be added all at once at the beginning, such as as iron sulfate. It is also effective to add more if the divalent iron ions added during the reaction are oxidized or precipitated and reduced in quantity. Note that if the hydroxylase of the present invention already contains a sufficient amount of divalent iron ions, it is not necessarily required to add them.

[0147] Furthermore, the contact is preferably carried out in the presence of L-ascorbic acid. The L-ascorbic acid is preferably used in the reaction solution at a concentration of typically 0.0001 mol / L to 0.2 mol / L, preferably 0.001 mol / L to 0.1 mol / L. Adding L-ascorbic acid can reduce the oxidation of divalent iron ions.

[0148] Furthermore, the contact is preferably carried out in the presence of citric acid. The citric acid is preferably used in the reaction solution at a concentration of typically 0.001 mol / L to 0.1 mol / L, preferably in the range of 0.005 mol / L to 0.02 mol / L.

[0149] The generated hydroxypipecolic acid can be purified by separating bacterial cells and proteins from the reaction solution using separation or purification methods known to those skilled in the art, such as centrifugation or membrane treatment, and then appropriately combining extraction with organic solvents such as 1-butanol and tert-butanol, distillation, column chromatography using ion exchange resins or silica gel, crystallization at the isoelectric point, or crystallization with monohydrochloride, dihydrochloride, calcium salt, etc.

[0150] In the method for producing hydroxypipecolic acid of the present invention, when using a reaction solution containing αKG obtained by contacting L-Glu with GOX or the like of the present invention, or a reaction solution containing αKG obtained by crudely purifying the said reaction solution, the reaction of contacting L-Glu with GOX or the like of the present invention and the reaction of contacting pipecolic acid with hydroxylase or the like may be carried out separately, continuously, or in parallel in the same reaction solution.

[0151] From the standpoint of manufacturing costs and reaction efficiency, it is desirable to carry out the reaction of contacting L-Glu with the GOX of the present invention and the reaction of contacting pipecolic acid with hydroxylase, etc., sequentially or in the same reaction solution.

[0152] Furthermore, when using a culture medium containing the enzyme obtained by culturing a microorganism or cell capable of producing the enzyme, a processed product of said microorganism or cell, and / or said microorganism or cell (for example, a transformant having DNA encoding pipecolic acid hydroxylase), different transformants may be used for pipecolic acid hydroxylase, GOX of the present invention, and catalase, or a transformant having DNA encoding two or more of pipecolic acid hydroxylase, GOX of the present invention, and catalase may be used. From the viewpoint of manufacturing cost, it is more desirable to use a transformant having DNA encoding all of pipecolic acid hydroxylase, GOX of the present invention, and catalase.

[0153] Furthermore, in the method for producing hydroxypipecolic acid of the present invention, the pipecolic acid may be purified or a solution containing pipecolic acid obtained by a microbial reaction.

[0154] When pipecolic acid is obtained from L-lysine by a bacterial reaction, the solution contains equimolar amounts of ammonium ions with respect to L-lysine. This is because ammonia is produced during the conversion of L-lysine to pipecolic acid, and this ammonia exists as ammonium ions in the reaction solution. Therefore, if the inhibitory effect of GOX activity on ammonium ions is high, αKG may not be produced, or the amount produced may be insufficient, even when using a solution containing pipecolic acid obtained by a bacterial reaction.

[0155] However, the GOX of the present invention has a low inhibitory effect on ammonium ions, and by using the GOX of the present invention, the production of αKG from a solution containing pipecolic acid obtained by a microbial reaction, and the subsequent production of hydroxypipecolic acid from pipecolic acid, can be carried out efficiently.

[0156] In the present invention, in order to produce αKG from L-Glu, it is desirable to add L-Glu in an equimolar or greater amount than pipecolic acid, and particularly preferably in an equimolar to 2x molar range.

[0157] The present invention's method for producing hydroxypipecolic acid makes it possible to industrially produce hydroxypipecolic acid, which is useful as a pharmaceutical intermediate, with high productivity, low cost, and low price.

[0158] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0159] In the examples, amino acids may be represented using the abbreviations shown in Table 2.

[0160]

[0161] In the examples, for example, the notation "F5Y" means the substitution of the fifth phenylalanine with tyrosine in a given amino acid sequence.

[0162] Furthermore, in the examples, "w / v%" means "weight / volume%".

[0163] Example 1: Preparation of ScGOX and ScGOX monoexpression plasmids Synthetic DNA (SEQ ID NO: 1) of the gene sequence scGOX (hereinafter sometimes referred to as "ScGOX") (SEQ ID NO: 2), which encodes a protein obtained by removing 18 amino acid residues from the N-terminus of the NAD(P)-binding protein derived from Streptomyces chattanoogenesis (GeneBank Access No. WP_053923987.1) and substituting the 19th amino acid residue, alanine (A), with methionine (M), was purchased from ATUM Corporation.

[0164] Using this synthetic DNA as a template, PCR was performed according to the attached protocol to obtain DNA fragments with restriction enzyme MfeI cleavage sites added to the 5' end and restriction enzyme XbaI cleavage sites added to the 3' end.

[0165] The obtained scgox DNA fragment was conjugated to a pKW32 plasmid (plasmid described in Japanese Patent Publication No. 5613660) digested with restriction enzymes MfeI and XbaI using T4DNA Ligase (Takara Bio Inc.), and a plasmid was prepared in which the scgox gene was introduced downstream of the trc promoter of the pKW32 plasmid. The base sequence of the obtained plasmid was confirmed and it was named pKW32-scgox.

[0166] Furthermore, plasmid pKW32-slgox, shown in Table 3, was also prepared using FAD-binding protein derived from Streptomyces lydicus (GeneBank Accession No. WP_069570642.1) in the same manner as described above.

[0167]

[0168] Reference Example 1: Preparation of Single Expression Plasmids of SxGOX and KateE pKW32-sxgox, shown in Table 4, was obtained in the same manner as in Example 1, except that the synthetic DNA used was the synthetic DNA of the gene sequence sxgox (Sequence ID 5 in this specification) encoding a protein (hereinafter sometimes referred to as "SxGOX") (Sequence ID 6 in this specification) in which the first amino acid residue at the N-terminus of GOX (amino acid sequence shown in Sequence ID 1 of Japanese Patent Publication No. 4002765) derived from Streptomyces sp. X-119-6 was replaced with methionine (M) (Sequence ID 6 in this specification). Furthermore, pKW32-kateE, shown in Table 4, was prepared using the same method as above for the catalase protein (GeneBank Accession No. YP_025308.1) derived from Escherichia coli K-12 MG1655 strain.

[0169]

[0170] Reference Example 2: Creation of a single-expression plasmid of hydroxylase McPH A plasmid (pJ411-McPH-m3a) containing a McPH gene was created by introducing three mutations, F5Y, C23A, and D282E, into the wild-type McPH gene encoded in the pJ411 plasmid, using the same method as in the example in Patent Document 6 (WO2017 / 057730).

[0171] Using pJ411-McPH-m3a as a template, PCR was performed according to the attached protocol using primers of Sequence ID No. 19 and Sequence ID No. 20, and PrimeSTAR® Max DNA Polymerase (manufactured by Takara Bio Inc.) to obtain a DNA fragment containing the McPH-m3a gene.

[0172] On the other hand, the pKW32 plasmid was cleaved with restriction enzymes MfeI and XbaI, subjected to 1 w / v% agarose gel electrophoresis, and the cleaved fragments were extracted from the agarose gel using the GEL / PCR Purification Mini Kit (Chiyoda Science Co., Ltd.) according to the attached protocol and purified.

[0173] The two DNA fragments obtained above were combined using the In-Fusion® HD Cloning Kit (Takara Bio Inc.) according to the attached protocol, and Escherichia coli JM109 (Takara Bio Inc.) was transformed using the resulting DNA solution according to the attached protocol. Furthermore, a plasmid was prepared by performing the miniprep method using the QIAprep® Spin Miniprep Kit (QIAGEN Inc.) according to the attached protocol. The base sequence of the obtained plasmid was confirmed and named pKW32-McPH-m3a.

[0174] Using the obtained pKW32-McPH-m3a as a template, DNA fragments were amplified by PCR using primers SEQ ID NO: 21 and SEQ ID NO: 22, and PrimeSTAR® Max DNA Polymerase (Takara Bio Inc.) according to the attached protocol. The resulting amplified solution was treated with the restriction enzyme DpnI at 37°C for 1.5 hours to cleave the template plasmid. Using the obtained DNA solution, Escherichia coli JM109 (Takara Bio Inc.) was transformed in the same manner as above, and plasmids were prepared by miniprep in the same manner as above. The base sequence of the obtained plasmid was confirmed and named pKW32-McPH-m4a.

[0175] Furthermore, plasmids shown in Table 5 were prepared using the templates and primer sets shown in Table 5 in the same manner as described above.

[0176]

[0177] Using the obtained pKW32-McPH-m4a as a template, PCR was performed in the same manner as described above, according to the attached protocol, using primers of SEQ ID NO: 31 and SEQ ID NO: 32, to obtain a DNA fragment containing the McPH gene.

[0178] On the other hand, using the pET24a vector as a template, PCR was performed in the same manner as above, according to the attached protocol, using primers of SEQ ID NO: 33 and SEQ ID NO: 34, to obtain DNA fragments containing the pET24a vector.

[0179] The two DNA fragments, one containing the McPH gene and the other containing the pET24a vector, were joined using the In-Fusion® HD Cloning Kit (Takara Bio Inc.) in the same manner as described above. Escherichia coli JM109 (Takara Bio Inc.) was transformed using the resulting DNA solution in the same manner as described above, and a plasmid was prepared by miniprep using the same method as described above. The base sequence of the obtained plasmid was confirmed and it was named pET-McPH-m4a.

[0180] Using the obtained pET-McPH-m4a as a template, PCR was performed according to the attached protocol using primers of SEQ ID NO: 35 and SEQ ID NO: 36, and PrimeSTAR® Max DNA Polymerase (manufactured by Takara Bio Inc.) to obtain DNA fragment No. 1.

[0181] Furthermore, using the templates and primer sets shown in Table 6, the PCR method was performed in the same manner as described above to obtain DNA fragments No. 2 to 5.

[0182] All of the obtained DNA fragments No. 1 to 5 were conjugated using Gibson Assembly Master Mix (New England Biolabs) according to the attached protocol. Escherichia coli DH5α (Takara Bio Inc.) was transformed using the obtained DNA solution in the same manner as described above. The cells were then inoculated into LB liquid medium containing kanamycin as an antibiotic. Plasmids were prepared from the obtained bacteria using the miniprep method in the same manner as described above, and a pET plasmid-type mutant library (pET-McPH-cmb-lib) (Table 7) was created.

[0183] Furthermore, a pKW plasmid-type mutant library (pKW-McPH-cmb-lib) (Table 7) was prepared in the same manner as described above, except that pKW32-McPH-m5c was used as the template instead of pET-McPH-m4a.

[0184] From the obtained pET plasmid mutant library, we obtained the plasmid pET24a-McPH-m7a, which encodes McPH-m7a (amino acid sequence: SEQ ID NO: 10, nucleic acid sequence: SEQ ID NO: 9), a McPH mutant with high pipecolic acid hydroxylation activity.

[0185] Furthermore, from the pKW plasmid mutant library, we obtained the plasmid pKW32-McPH-m7e, which encodes McPH-m7e (amino acid sequence: SEQ ID NO: 12, nucleic acid sequence: SEQ ID NO: 11), a McPH mutant with high pipecolic acid hydroxylation activity.

[0186]

[0187]

[0188] Using the obtained plasmid pET-McPH-m7a as a template, a portion of the McPH gene was amplified by PCR using primers of SEQ ID NO: 49 and SEQ ID NO: 50 according to the attached protocol. Furthermore, using plasmid pKW32-McPH-m6a as a template, PCR was performed using primers of SEQ ID NO: 51 and SEQ ID NO: 52 according to the attached protocol to obtain a DNA fragment containing the vector portion of the McPH gene of plasmid pKW32-McPH-m6a, including the N-terminus and C-terminus.

[0189] The two obtained DNA fragments were joined using Gibson Assembly Master Mix (New England Biolabs) according to a standard protocol. Escherichia coli JM109 (Takara Bio Inc.) was transformed using the resulting DNA solution in the same manner as described above, and a plasmid was prepared by miniprep using the same method as described above. The base sequence of the obtained plasmid was confirmed and named pKW-McPH-m7a.

[0190] Example 2: Preparation of a plasmid for co-expression of GOX and catalase according to the present invention. The pKW32-scgox obtained in Example 1 was digested with restriction enzymes SpeI and NdeI according to the attached protocol to obtain a gene fragment of scgox. Similarly, the pKW32-kateE obtained in Reference Example 1 was digested with restriction enzymes XbaI and NdeI according to the attached protocol to obtain a gene fragment containing pKW32-kateE.

[0191] The two gene fragments obtained, scGOX and pKW32-kateE, were ligated using T4 DNA Ligase (Takara Bio Inc.) to obtain the co-expression plasmid pKW32-kateE+scGOX, which expresses both ScGOX and catalase.

[0192] Furthermore, using pKW32-slgox, a co-expression plasmid of SlGOX and catalase, pKW32-katE+slgox, was obtained in the same manner as described above.

[0193] Reference Example 3: Preparation of a plasmid for co-expression of SxGOX and catalase The SxGOX and catalase co-expression plasmid pKW32-katE+sxgox was obtained in the same manner as in Example 2, except that pKW32-sxgox was used instead of pKW32-scgox.

[0194] Example 3: Preparation of a plasmid for co-expression of ScGOX, McPH-m7e, and catalase. The pKW32-katE+scgox obtained in Example 2 was digested with restriction enzymes SpeI and NdeI according to the attached protocol to obtain a gene fragment containing katE+scgox. Similarly, the pKW32-McPH-m7e obtained in Reference Example 2 was digested with restriction enzymes XbaI and NdeI according to the attached protocol to obtain a gene fragment containing pKW32-McPH-m7e.

[0195] The two obtained gene fragments were ligated using T4 DNA Ligase (Takara Bio Inc.) to obtain pKW32-McPH-m7e+kateE+scgox.

[0196] Reference Example 4: Preparation of genetically modified E. coli expressing various enzymes The following procedure is used when expressing enzymes in E. coli, specifically when co-expressing GOX of the present invention, GOX of the comparative example, hydroxylase, or two or more of these.

[0197] It should be noted that there are other known methods and conditions for creating recombinant E. coli besides the procedure described below. This invention is not limited to the procedure described below.

[0198] (1) Preparation of genetically modified Escherichia coli Using the plasmids obtained in Examples 1 to 3 or Reference Examples 1 to 3, Escherichia coli JM109 competent cell (manufactured by Takara Bio Inc.) was transformed according to the attached protocol to obtain recombinant Escherichia coli into which each plasmid was introduced.

[0199] (2) Flask culture The recombinant Escherichia coli obtained in (1) above was inoculated into LB liquid medium containing 25 mg / L kanamycin sulfate and 0.2 mmol / L isopropyl-β-thiogalactopyranoside (hereinafter sometimes referred to as "IPTG") as antibiotics, and cultured in a flask at 30°C for 18 hours with shaking. The bacterial cells were collected from the resulting culture solution by centrifugation.

[0200] (3) Jar culture Recombinant Escherichia coli obtained in (1) above was inoculated into LB liquid medium containing 25 mg / L kanamycin sulfate as an antibiotic and cultured in a flask at 30°C for 18 hours with shaking. The resulting culture solution was mixed with 2 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 5 g / L dipotassium hydrogen phosphate, 5 g / L yeast extract (Meat P1G (Asahi Food and Healthcare Co.)), 5 g / L Sollis 095E (ROQUETTE Co.), 100 g / L glycerol, 0.1 g / L magnesium sulfate heptahydrate, and 0.084 g / L iron sulfate heptahydrate. The bacteria were inoculated at 2% by volume into a liquid culture medium containing 6 mg / L copper sulfate pentahydrate, 8.4 mg / L zinc sulfate heptahydrate, 7.2 mg / L cobalt nitrate hexahydrate, 6 mg / L disodium molybdate dihydrate, 2.3 mg / L calcium chloride, 1.5 mg / L boric acid, 0.2 g / L adecanol LG-109 (manufactured by ADEKA Corporation), and 25 mg / L kanamycin sulfate.

[0201] The liquid culture medium inoculated with the above-mentioned bacteria was maintained at a culture temperature of 30°C, a dissolved oxygen concentration of 20%, a pH of 7.0 (adjusted with 14 w / v% ammonia water), and an aeration rate of 1:1 volume of air per minute relative to the volume of the culture medium. Nine hours after inoculation, IPTG was added to a concentration of 0.2 mmol / L, and the culture was incubated overnight. The bacterial cells were collected from the resulting culture solution by centrifugation.

[0202] Reference Example 5: Preparation of Enzyme Solution In this example, enzyme solutions were obtained by appropriately adopting the enzyme solution preparation methods described in (1), (2), or (3) below, and these were used for reaction and activity measurements.

[0203] It should be noted that there are other known methods and conditions for preparing enzyme solutions besides the one described below. This invention is not limited to the preparation method described below.

[0204] (1) The bacterial cells obtained in Reference Example 4 of surfactant treatment were suspended in 0.1 w / v% Triton-(registered trademark) X-100 (manufactured by Sigma-Aldrich) at concentrations of 10 g / L, 20 g / L, 100 g / L, or 500 g / L to prepare an enzyme solution.

[0205] (2) The bacterial cells obtained in Reference Example 4 of the enzyme treatment were suspended in a bacterial cell lysis buffer (100 mmol / L of 2-morpholinoethanesulfonic acid (MES) buffer (pH 7.0), 2 mmol / L of MgSO4, 0.1 w / v% of Triton® X-100 (manufactured by Sigma-Aldrich) and a lysozyme solution (50 mmol / L of MES buffer (pH 7.0), 100 mmol / L of NaCl, 10 mg / mL of lysozyme, 1 mg / mL of DNaseI (manufactured by Worthington Biochemicals), and 50% glycerol) with 1 / 50th volume (molar ratio) added, and the cells were lysed by shaking at 20°C for 10 minutes. The obtained bacterial cell lysis solution was centrifuged, and the supernatant was used as the enzyme solution.

[0206] (3) The bacterial cells obtained in Reference Example 4 of the ultrasonic disruption treatment were suspended in distilled water to a concentration of 1 g / L, ultrasonic disruption was performed for 15 minutes, and then the cells were centrifuged. The supernatant was used as the enzyme solution.

[0207] Reference Example 6: HPLC Analysis In this example, enzyme solutions were obtained by appropriately employing the enzyme solution preparation methods described in (1), (2), or (3) below, and these were used for reaction and activity measurements.

[0208] In this example, analysis was performed by high-performance liquid chromatography (HPLC) as described in (1) or (2) below to measure the concentrations of αKG, L-Glu, L-pipecolic acid (hereinafter sometimes referred to as "LPA"), or cis-5-hydroxy-L-pipecolic acid (hereinafter sometimes referred to as "5HPA").

[0209]

[0210]

[0211] Reference Example 7: Measurement of αKG concentration by absorbance measurement 10 mmol / L L-pipecolic acid, αKG (appropriate concentration from 0 to 10 mmol / L), 1 mmol / L L-ascorbic acid, 10 mmol / L DTT, 0.5 mmol / L FeSO ４To 18 μL of αKG measurement solution containing 2 mmol / L sodium citrate and 100 mmol / L MES buffer (pH 7.0), 100 μL of semicarbazide hydrochloride (SCA) solution (10 g / L SCA, 15 g / L sodium acetate) was added. Then, 2 μL of enzyme solution prepared from recombinant pKW32-McPH-m3a Escherichia coli according to the procedure of Reference Example 5(3) was added to each solution. After standing at 37°C for 30 minutes, the absorbance at a wavelength of 250 nm was measured using a microplate reader (Molecular Bio).

[0212] Figure 1 shows the relationship between the αKG concentration (in the range of 0 to 10 mmol / L) in the αKG measurement solution and the absorbance at a wavelength of 250 nm.

[0213] As is clear from Figure 1, a linear correlation was observed between the αKG concentration in the αKG measurement solution and the absorbance at a wavelength of 250 nm. This suggests that the αKG concentration can be estimated by measuring the absorbance at a wavelength of 250 nm of the αKG measurement solution containing SCA.

[0214] Example 4: Confirmation of GOX activity inhibition by αKG Using pKW32-katE+scgox and pKW32-katE+slgox obtained in Example 2, and pKW32-katE+sxgox obtained in Reference Example 3, bacterial cells were obtained according to the procedures of Reference Example 4 and Reference Example 5, respectively, and the respective enzyme solutions were obtained according to the procedure of Reference Example 5(1). The relative activity of each enzyme solution was measured as follows.

[0215] In a 2 mL microtube, 5 μL of 2 mol / L L-sodium glutamate and 10 μL of the enzyme solution obtained above were added. 2 mol / L αKG aqueous solution (pH adjusted to 7 with sodium hydroxide) and distilled water were added to prepare 100 μL of reaction solution, resulting in αKG concentrations of 0 mmol / L, 200 mmol / L, 400 mmol / L, and 800 mmol / L, respectively. This reaction solution was then incubated at a reaction temperature of 15°C with sufficient stirring speed for 30 minutes.

[0216] After the reaction, 50 μL of 1 mol / L hydrochloric acid was added to the reaction solution to stop the reaction, then 50 μL of 1 mol / L sodium hydroxide aqueous solution was added to neutralize it, and 2 mmol / L CuSO4 was added. ４ 200 μL of the solution was added and thoroughly mixed. The supernatant was obtained by centrifugation and analyzed using HPLC condition 1 to quantify the L-Glu concentration.

[0217] Relative activity was calculated from the decrease in L-Glu. One unit of activity was defined as the decrease of 1 μmol of L-Glu (production of αKG) per minute at 15°C. Using the activity at an αKG concentration of 0 mmol / L as the baseline, the relative activity was calculated for αKG concentrations of 200 mmol / L, 400 mmol / L, and 800 mmol / L. The results are shown in Figure 2.

[0218] As is clear from Figure 2, under conditions of an αKG concentration of 800 mmol / L, the relative activity of ScGOX and SlGOX remained higher (over 70%) than that of the comparative example SxGOX, with ScGOX showing a relative activity of 120%. From this, it can be seen that even under high αKG concentration conditions, ScGOX and SlGOX are less affected by or unaffected by αKG-induced product inhibition (GOX activity inhibition), and their GOX activity is maintained.

[0219] Example 5: Confirmation of GOX activity inhibition by ammonium ions Using pKW32-katE+scgox and pKW32-katE+slgox obtained in Example 2, and pKW32-katE+sxgox obtained in Reference Example 3, bacterial cells were obtained according to the procedures of Reference Example 4 and Reference Example 5, respectively, and the respective enzyme solutions were obtained according to the procedure of Reference Example 5(1). The relative activity of each enzyme solution was measured as follows.

[0220] In a 2 mL microtube, add 0.5 μL of 2 mol / L L-sodium glutamate and 10 μL of the enzyme solution. Add 2 mol / L ammonium chloride (hereinafter referred to as "NH") to achieve concentrations of 0 mmol / L, 100 mmol / L, 200 mmol / L, 400 mmol / L, or 600 mmol / L. ４Sometimes referred to as "Cl," ) and distilled water were added to prepare 100 μL of reaction solution. This reaction solution was reacted for 60 minutes at a reaction temperature of 15°C with a sufficient stirring speed.

[0221] After the reaction, the αKG concentration was measured using the method described in Reference Example 7. Specifically, the absorbance of the obtained reaction solution at 250 nm was measured, and the amount of αKG was calculated from a calibration curve previously prepared showing the relationship between absorbance and αKG amount. One unit was defined as the activity that produces αKG from 1 μmol of L-Glu per minute at 15°C. NH ４ Using the activity at a Cl concentration of 0 mmol / L as a reference, each NH ４ The relative activity of each compound at different Cl concentrations was calculated. The results are shown in Figure 3.

[0222] As is clear from Figure 3, NH ４ Under conditions where the Cl concentration was 200 mmol / L to 600 mmol / L, the relative activity of ScGOX and SlGOX remained higher than that of the comparative example SxGOX, and in particular, ScGOX showed higher relative activity than NH ４ Relative activity of 100% or more was observed at all Cl concentrations from 200 mmol / L to 600 mmol / L. This indicates that ScGOX and SlGOX maintain their GOX activity even under high ammonium ion concentration conditions, with little to no influence from product inhibition (GOX activity inhibition) by ammonium ions.

[0223] Reference Example 8: Preparation of L-pipecolic acid reaction solution (1) Preparation of plasmids for expression of L-pipecolic acid synthases Synthetic DNA of the gene sequence dpkA encoding N-methylamino acid dehydrogenase (SEQ ID NO: 14) derived from Pseudomonas ptyida ATCC12633 strain (SEQ ID NO: 13) (SEQ ID NO: 13) was purchased from ATUM Corporation.

[0224] Using this synthetic DNA as a template, PCR was performed according to the attached protocol to amplify DNA fragments in which the restriction enzyme EcoRI site was attached to the 5'- side of dpkA and the restriction enzyme XbaI site was attached to the 3'- side.

[0225] The obtained DNA fragment was conjugated to a pKW32 plasmid digested with restriction enzymes MfeI and XbaI using T4DNA Ligase (Takara Bio Inc.), and a plasmid was prepared by introducing the dpkA gene downstream of the trc promoter of the pKW32 plasmid. The base sequence of the obtained plasmid was confirmed and named pKW32-dpkA.

[0226] Furthermore, the plasmids pKW32-dkdh and pKW32-lysR shown in Table 8 were prepared in the same manner as described above for the synthetic DNA (SEQ ID NO: 15) of the gene sequence dkdh encoding D-amino acid dehydrogenase (SEQ ID NO: 16) from Selenomonas ruminantium subsp. lactilytica TAM6421, and for the synthetic DNA (SEQ ID NO: 17) of the gene sequence lysR encoding a lysine racemase mutant (SEQ ID NO: 18) from Pseudomonas putida KT2440, respectively.

[0227] Furthermore, using the three plasmids shown in Table 8, three gene co-expression plasmids pKW32-dpkA+dkdh+lysR were obtained in the same manner as in Example 2.

[0228]

[0229] (2) Preparation of L-pipecolic acid reaction solution Using the pKW32-dpkA+dkdh+lysR obtained in (1) above, bacterial cells expressing the three enzymes were obtained according to the procedure of Reference Example 4.

[0230] 58.5 g of L-lysine hydrochloride and 0.12 g of nicotinamide adenine dinucleotide phosphate (oxidized form) were added to a flask, and the mixture was adjusted to 0.38 L and pH 9.0 by adding water and sodium hydroxide solution. To this, 0.02 L of a suspension of the bacterial cells obtained above in water at 50 g / L was added, and the mixture was reacted for 40 hours at 30°C.

[0231] The resulting reaction solution was analyzed for LPA concentration using HPLC conditions 1 of Reference Example 6, and the result showed an LPA concentration of 952 mmol / L. Furthermore, since the reaction from L-Lys to LPA produces an equimolar amount of ammonia along with LPA, it is considered that the resulting L-pipecolic acid reaction solution contains ammonium ions at approximately the same molar concentration as LPA.

[0232] Example 6: Confirmation of GOX activity in hydroxylation reaction substrate solution Using pKW32-katE+scgox and pKW32-katE+slgox obtained in Example 2, and pKW32-katE+sxgox obtained in Reference Example 3, bacterial cells were obtained according to the procedure of Reference Example 4, and enzyme solutions were obtained according to the procedure of Reference Example 5(1).

[0233] Using the enzyme solutions described above, the GOX activity of ScGOX, SLGOX, and SXGOX was measured in the presence of a hydroxylation reaction substrate solution as follows.

[0234] Specifically, 50 μL of the L-pipecolic acid reaction solution prepared in Reference Example 8(2), 9.4 mg of L-sodium glutamate monohydrate, 2.5 μL of 2 mol / L L-sodium ascorbate aqueous solution, 0.5 μL of 100 mmol / L ferrous sulfate aqueous solution, and 10 μL of 100 mmol / L sodium citrate aqueous solution were added to a 2 mL microtube. After adjusting the pH to 7 with 1 mol / L hydrochloric acid, distilled water was added to prepare a 90 μL substrate solution.

[0235] To this substrate solution, 10 μL of the enzyme solution prepared from 20 g / L bacterial cells was added, and the reaction was carried out at a reaction temperature of 15°C with a sufficient stirring speed for 60 minutes.

[0236] Furthermore, for baseline measurement in HPLC analysis, 0.1 w / v% Triton®-X-100 (manufactured by Sigma-Aldrich) was added to the substrate solution instead of the enzyme solution, and the reaction was carried out for 60 minutes at a reaction temperature of 15°C with sufficient stirring speed, as described above.

[0237] Add 0.108% HClO to each of the resulting reaction solutions. ４300 μL was added, the supernatant was obtained by centrifugation, and the analysis was performed under HPLC conditions 2 of Reference Example 6. The amount of αKG produced was quantified by the peak area obtained by subtracting the peak area that appears at the same position as αKG in the baseline measurement sample from the peak area of ​​αKG, and the GOX activity was calculated. One unit was defined as the activity that produces αKG from 1 μmol of L-Glu per minute at 15°C.

[0238] Next, the GOX activity was measured in the absence of the hydroxylation reaction substrate solution.

[0239] Specifically, 5 μL of 2 mol / L L-sodium glutamate aqueous solution and 75 μL of water were added to a 2 mL microtube to prepare an 80 μL reaction solution. Then, 20 μL of 10 g / L enzyme solution was added, and the reaction and HPLC analysis were performed in the same manner as described above to calculate the GOX activity.

[0240] For the GOX activity of the obtained ScGOX, SlGOX, and SxGOX, the relative activity of the GOX activity with the hydroxylation reaction substrate solution was calculated, using the GOX activity without the hydroxylation reaction substrate solution as the baseline. The results are shown in Figure 4.

[0241] As is clear from Figure 4, when the hydroxylation reaction substrate solution is included, the relative activity of the comparative example SxGOX decreases significantly to below 30%, while ScGOX and SlGOX maintain high relative activity, with ScGOX maintaining particularly high relative activity.

[0242] Here, since the hydroxylation reaction substrate solution contains ammonium ions derived from the L-pipecolic acid reaction solution, the decrease in the relative activity of SxGOX is thought to be due to product inhibition (GOX activity inhibition) by ammonium ions.

[0243] From this, it is thought that if the αKG production reaction and the LPA hydroxylation reaction are attempted to be carried out in parallel in the same reaction solution, the GOX activity may decrease with SxGOX and the reaction may not proceed, whereas ScGOX and SlGOX maintain high GOX activity, making it possible for the reactions to proceed simultaneously.

[0244] Example 7: Confirmation of αKG production by GOX in a hydroxylation reaction substrate solution Using pKW32-katE+scgox and pKW32-katE+slgox obtained in Example 2, bacterial cells were obtained according to the procedure of Reference Example 4, and the respective enzyme solutions were obtained according to the procedure of Reference Example 5(1). The αKG concentration of each enzyme solution was measured as follows.

[0245] In a 1 L jar fermenter (manufactured by Biot), 150 mL of the L-pipecolic acid reaction solution obtained in Reference Example 8 (2), 28.1 g of L-sodium glutamate monohydrate, 3.0 g of L-sodium ascorbate, 41.7 mg of iron(II) sulfate heptahydrate, and 0.6 g of citric acid monohydrate were added. The pH was adjusted to 7 with 1 mol / L hydrochloric acid, and then distilled water was added to prepare a 270 mL substrate solution. To this substrate solution, 30 mL of the enzyme solution prepared from 100 g / L of bacterial cells was added, and the reaction was carried out for 63 hours under the conditions of a reaction temperature of 15°C, an aeration rate of 0.4 L / min, and a stirring speed of 900 rpm. During the reaction, if foam increased, the defoaming agent LG109 was added as appropriate.

[0246] The reaction solution was sampled as needed during the reaction, and the resulting solution was mixed with 0.108% HClO. ４ After appropriate dilution, the supernatant was obtained by centrifugation, and the αKG concentration was measured under HPLC conditions 2 of Reference Example 6. The measurement results of the αKG concentration are shown in Figure 5.

[0247] As is clear from Figure 5, both SlGOX and ScGOX showed a time-dependent increase in αKG concentration even in the hydroxylation reaction substrate solution. In particular, with ScGOX, the αKG concentration reached 460 mmol / L 15 hours after the start of the reaction, and the molar yield from the amount of L-Glu used in the reaction was a high 92%.

[0248] Example 8: Parallel reactions of αKG production reaction and LPA hydroxylation reaction in the same reaction solution - 1 Using pKW32-katE+scgox and pKW32-katE+slgox obtained in Example 2, and pKW32-katE+sxgox obtained in Reference Example 3, bacterial cells were obtained according to the procedure of Reference Example 4, and enzyme solutions containing each GOX and catalase were obtained according to the procedure of Reference Example 5(1). Similarly, using pKW32-McPH-m7a obtained in Reference Example 2, an enzyme solution containing the hydroxylase McPH-m7a was obtained. For each enzyme solution, the αKG production reaction and the LPA hydroxylation reaction were confirmed as follows.

[0249] In a 1 L jar fermenter (manufactured by Biot), 150 mL of the L-pipecolic acid reaction solution obtained in Reference Example 8 (2), 28.1 g of L-sodium glutamate monohydrate, 3.0 g of L-sodium ascorbate, 41.7 mg of iron(II) sulfate heptahydrate, and 0.6 g of citric acid monohydrate were added. The pH was adjusted to 7 with 1 mol / L hydrochloric acid, and then distilled water was added to prepare a 230 mL substrate solution. To this substrate solution, 30 mL of enzyme solution containing GOX and catalase prepared from 100 g / L bacterial cells, and 40 mL of enzyme solution containing 500 g / L hydroxylase McPH-m7a were added. The reaction was then carried out for 63 hours under the conditions of a reaction temperature of 15°C, an aeration rate of 0.4 L / min, and a stirring speed of 900 rpm. During the reaction, if foam increased, the defoaming agent LG109 was added as appropriate.

[0250] The reaction solution was sampled as needed during the reaction, and the reaction was stopped by adding 0.5 times the volume of 1 mol / L hydrochloric acid to the obtained solution. Then, 1 mol / L aqueous sodium hydroxide solution was added. The resulting solution was then dissolved in 2 mmol / L CuSO4. ４ After diluting 20-fold with an aqueous solution, the supernatant was obtained by centrifugation, and L-Glu, LPA, and 5HPA were measured under HPLC conditions 1 of Reference Example 6.

[0251] The conversion rate from LPA to 5HPA in the LPA hydroxylation reaction was calculated using the formula "Conversion Rate [%] = 5HPA concentration / (LPA concentration + 5HPA concentration)" based on the LPA and 5HPA concentrations obtained by analysis. Figure 6(1) shows the L-Glu concentration, and Figure 6(2) shows the conversion rate results.

[0252] As is clear from Figure 6, both SlGOX and ScGOX reached a conversion rate of 100% in 63 hours, completely completing the conversion from LPA to 5HPA, whereas the conversion rate of the comparative example, SxGOX, was less than 15%. Furthermore, even after 63 hours from the start of the reaction, the L-Glu concentration of SxGOX had only decreased by 100 mmol / L. This suggests that the LPA hydroxylation reaction was not proceeding due to the low rate of αKG production from L-Glu by SxGOX. On the other hand, the L-Glu concentration of both SlGOX and ScGOX decreased linearly from the start of the reaction, confirming that αKG production from L-Glu was proceeding continuously and at a rapid rate.

[0253] Thus, when the αKG production reaction and the LPA hydroxylation reaction are carried out in parallel in the same reaction solution, it is clear that the reaction can be carried out efficiently by using ScGOX or SLGOX of the present invention.

[0254] Example 9: Parallel reactions of αKG production reaction and LPA hydroxylation reaction in the same reaction solution - 2 Using pKW32-McPH-m7e+katE+scgox obtained in Example 3, bacterial cells were obtained according to the procedure of Reference Example 4, and the enzyme solution was obtained according to the procedure of Reference Example 5(1).

[0255] Next, 200 mL of the L-pipecolic acid reaction solution obtained in Reference Example 8(2), 37.4 g of L-sodium glutamate monohydrate, 4.0 g of L-sodium ascorbate, 55.6 mg of iron(II) sulfate heptahydrate, and 0.8 g of citric acid monohydrate were added to a 1 L jar fermenter (manufactured by Biot). The pH was adjusted to 7 with 1 mol / L hydrochloric acid, and then distilled water was added to prepare a 346 mL substrate solution. 54 mL of the enzyme solution prepared from 100 g / L of bacterial cells was added to the obtained substrate solution, and the reaction was carried out for 43 hours under the conditions of a reaction temperature of 15°C, an aeration rate of 0.4 L / min, and a stirring speed of 900 rpm. During the reaction, if foam increased, the defoaming agent LG109 was added as appropriate.

[0256] The reaction solution was sampled as needed during the reaction and analyzed by HPLC in the same manner as in Example 8 to calculate the conversion rate from LPA to 5HPA. The results are shown in Figure 7.

[0257] As is clear from Figure 7, when bacterial cells expressing ScGOX, catalase, and hydroxylase McPH-m7e simultaneously were used, the conversion rate reached 100% in 43 hours, indicating that the conversion from LPA to 5HPA was completely completed.

[0258] This confirms that, in addition to the method using two types of bacterial cells expressing GOX and catalase and hydroxylase, as in Example 8, parallel reactions of the αKG production reaction and the LPA hydroxylation reaction can be carried out in the same reaction solution when using a bacterial cell that simultaneously expresses GOX, catalase, and hydroxylase in a single type of bacterial cell, as in Example 9.

Claims

1. A glutamate oxidase having a polypeptide comprising any of the following amino acid sequences: (1) the amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 2 or 4; (3) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4.

2. Nucleic acids encoding any of the following amino acid sequences: (1) the amino acid sequence represented by SEQ ID NO: 2 or 4; (2) an amino acid sequence having one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence represented by SEQ ID NO: 2 or 4; (3) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4.

3. The nucleic acid according to claim 2, comprising any of the following base sequences: (4) the base sequence represented by SEQ ID NO: 1 or 3; (5) the base sequence having one or more base substitutions, deletions, and / or additions in the base sequence represented by SEQ ID NO: 1 or 3; (6) the base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1 or 3.

4. A recombinant vector comprising the nucleic acid described in claim 2 or 3.

5. The recombinant vector according to claim 4, further comprising a nucleic acid encoding a hydroxylase and / or a nucleic acid encoding catalase.

6. A transformant comprising the recombinant vector described in claim 4.

7. A method for producing α-ketoglutaric acid, comprising contacting glutamic acid with glutamic acid, a culture medium containing glutamic acid oxidase as described in claim 1, a microorganism or cell capable of producing the glutamic acid oxidase, a processed product of the microorganism or cell, and / or the glutamic acid oxidase obtained by culturing the microorganism or cell.

8. The method for producing α-ketoglutaric acid according to claim 7, wherein the microorganism or cell is the transformant according to claim 6.

9. A method for producing hydroxypipecolic acid, comprising hydroxylating pipecolic acid with a hydroxylase in the presence of α-ketoglutaric acid, wherein α-ketoglutaric acid is obtained by the production method described in claim 7.

10. The method for producing hydroxypipecolic acid according to claim 9, wherein the microorganism or cell is a transformant that co-expresses the hydroxylase and the glutamate oxidase and / or catalase.

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