L-pipecolic acid hydroxylase and method for producing hydroxy-l-pipecolic acid

Abstract

The present invention addresses the problem of providing a stable hydroxylase having high L-pipecolic acid hydroxylation activity and high stability by introducing a mutation into an L-pipecolic acid hydroxylase.　It has been found that the effects of improving the L-pipecolic acid hydroxylation activity and stability of a hydroxylase (McPH) when the hydroxylase is expressed in a host such as E. coli can be obtained by identifying an amino acid residue that can affect an improvement in the hydroxylation activity and stability of the hydroxylase, and introducing a mutation into the identified amino acid residue.

Description

Method for producing L-pipecolic acid hydroxylase and hydroxy-L-pipecolic acid

[0001] The present invention relates to a biocatalyst that produces hydroxy-L-pipecolic acid by hydroxylating L-pipecolic acid, and a method for producing hydroxy-L-pipecolic acid using the same.

[0002] Hydroxy-L-pipecolic acid is a compound useful as an intermediate for synthesizing pharmaceuticals, etc. For example, it is known that (4S)-hydroxy-L-pipecolic acid can be used as a precursor of a Rho kinase inhibitor (Patent Document 1), (4R)-hydroxy-L-pipecolic acid can be used as a precursor of the HIV protease inhibitor parinavir (Non-Patent Document 1), and (5S)-hydroxy-L-pipecolic acid and (5R)-hydroxy-L-pipecolic acid can be used as precursors of antibacterial agents (Patent Document 2).

[0003] It is known that hydroxy-L-pipecolic acid can be produced from L-pipecolic acid by biological methods, for example, by BAB52605 protein derived from the Lotus japonicus root nodule bacterium Mesorhizobium loti MAFF303099, CAC47686 protein derived from the alfalfa root nodule bacterium Sinorhizobium meliloti 1021, or Segniliparus rugosus ATCC 104444. It has been reported that a protein encoded by a polynucleotide (cis gene) expressed from 48 bases (corresponding to 16 amino acids) upstream of the annotation of the EFV12517 protein derived from BAA-974 has the ability to convert L-pipecolic acid to (5S)-hydroxy-L-pipecolic acid (Patent Document 3), and L-pipecolic acid can be converted to (5S)-hydroxy-L-pipecolic acid using these hydroxylases.

[0004] On the other hand, the hydroxylases described in these patent documents that have the ability to convert L-pipecolic acid to hydroxy-L-pipecolic acid also have the ability to hydroxylate L-proline to produce hydroxy-L-proline.

[0005] Here, Patent Document 4 reports that the WP_030487089 protein (hereinafter, may be referred to as "McPH") derived from Micromonospora chokoriensis has a low activity of hydroxylating L-proline and can yield highly pure (5S)-hydroxy-L-pipecolic acid.

[0006] However, in order to produce pharmaceutical intermediates and the like industrially and more efficiently and inexpensively, a compound having even higher L-pipecolic acid hydroxylating activity is desired.

[0007] Furthermore, for industrial production, from the viewpoint of cost and availability, it is desirable to have an enzyme that exhibits high L-pipecolic acid hydroxylation activity when expressed in Escherichia coli as a host. While Escherichia coli typically grows well when cultured at temperatures of about 25°C to about 40°C, some hydroxylases may not exhibit sufficient activity or may be inactivated within this temperature range. Furthermore, when a reaction using a hydroxylase is attempted at a reaction temperature of about 25°C to about 50°C, which is known to be the optimum temperature for the enzyme, the hydroxylase may be inactivated within this temperature range.

[0008] Therefore, there is a demand for a hydroxylase with improved stability that is not inactivated even in reactions at such relatively high temperatures.

[0009] Furthermore, improving the stability of the hydroxylase improves the stability of the enzyme during cultivation and reaction, making it possible to extend the half-life of the enzyme activity decline, reduce the amount of enzyme used in reactions at high temperatures, and reduce the purification load for removing the enzyme after the reaction.

[0010] JP 2010-514720 A JP 2004-505088 A International Publication No. 2013 / 187438 Pamphlet International Publication No. 2017 / 057730 Pamphlet

[0011] Gillard et al. , The Journal of Organic Chemistry, 1996, Vol. 61, p. 2226

[0012] As described above, methods for producing hydroxy-L-pipecolic acid from L-pipecolic acid using a hydroxylase have been known, but have had problems such as low productivity and insufficient stability. Therefore, these methods are not satisfactory as methods for producing intermediates for pharmaceuticals and the like that require high purity. Therefore, there is a need for an efficient production method that can produce highly pure hydroxy-L-pipecolic acid at high productivity and low cost, and an L-pipecolic acid hydroxylase to be used therein.

[0013] The low productivity of hydroxy-L-pipecolic acid is thought to be due to, for example, low activity of the hydroxylase, low yield of the solubilized enzyme when expressed in a host such as E. coli, and tendency of the enzyme to be inactivated in the temperature range (e.g., about 25°C to about 50°C) during expression in a host such as E. coli or during the hydroxylation reaction. Therefore, there is a need for a highly stable hydroxylase that has high L-pipecolic acid hydroxylating activity and is not inactivated even in the relatively high temperature range during expression in a host such as E. coli or during the hydroxylation reaction.

[0014] An object of the present invention is to provide a stable hydroxylase having high L-pipecolic acid hydroxylating activity and high stability by introducing a mutation into L-pipecolic acid hydroxylase, and a further object of the present invention is to provide a method for industrially producing hydroxy-L-pipecolic acid from L-pipecolic acid with high productivity and at low cost using such a hydroxylase.

[0015] The present inventors conducted extensive research to solve the above-mentioned problems. As a result of extensive investigations, they performed sequence homology analysis with other hydroxylases on McPH, a hydroxylase described in Patent Document 4, and performed three-dimensional structural analysis of McPH by computer simulation. As a result, they identified amino acid residues that may affect the hydroxylation activity and stability of McPH. They then found that introducing mutations into the identified amino acid residues can improve the L-pipecolic acid hydroxylation activity and stability of the enzyme itself when the hydroxylase is expressed in a host such as Escherichia coli. Furthermore, they found that such an L-pipecolic acid hydroxylase can be used to produce hydroxy-L-pipecolic acid from L-pipecolic acid with high productivity (high optical purity and high concentration) at low cost and on an industrial scale. The present invention was achieved based on these findings.

[0016] That is, the gist of the present invention is as follows: [1] An L-pipecolic acid hydroxylase comprising a polypeptide having any of the following amino acid sequences (1) to (3): (1) an amino acid sequence represented by SEQ ID NO: 2 in which one or more of the 111th, 105th, 124th, 132nd, 140th, 249th, and 278th amino acids are substituted with other amino acids; (2) an amino acid sequence in which the amino acid sequence of (1) has amino acids other than the substituted amino acids modified, and which has 90% or more sequence identity with the amino acid sequence of (1); (3) an amino acid sequence in which the amino acid sequence of (1) has one to several amino acid substitutions, deletions, and / or additions other than the substituted amino acids. [1'] An L-pipecolic acid hydroxylase comprising a polypeptide having any of the following amino acid sequences (1') to (3'): (1') an amino acid sequence represented by SEQ ID NO: 2, in which the amino acid at position 111 has been substituted with another amino acid; (2') an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (1'), in which amino acids other than the substituted amino acid have been modified; (3') an amino acid sequence represented by (1'), in which the amino acid at position 111 has been substituted with another amino acid. [1''] The L-pipecolic acid hydroxylase according to [1'], which is the amino acid sequence of (1'), in which one or more amino acids at positions 105, 124, 132, 140, 249, and 278 have been substituted with another amino acid. [1'''] The L-pipecolic acid hydroxylase according to [1'], which is the amino acid sequence of (1') above, further comprising substitutions at the 105th and 124th positions with other amino acids. [1''''] The L-pipecolic acid hydroxylase according to [1'], which is the amino acid sequence of (1') above, further comprising substitutions at the 105th, 124th, and 249th positions with other amino acids.[2] The L-pipecolic acid hydroxylase according to [1], wherein the amino acid sequence of (1) is an amino acid sequence having one or more amino acid substitutions selected from the following amino acid substitutions (a) to (g) in the amino acid sequence of SEQ ID NO: 2: (a) substitution of isoleucine at position 105 with leucine; (b) substitution of leucine at position 111 with phenylalanine; (c) substitution of glutamic acid at position 124 with alanine; (d) substitution of alanine at position 132 with glutamine or lysine; (e) substitution of asparagine at position 140 with serine or methionine; (f) substitution of serine at position 249 with aspartic acid; (g) substitution of histidine at position 278 with tyrosine. [2'] The L-pipecolic acid hydroxylase according to [1'] to [1"'], wherein the amino acid sequence of (1') is an amino acid sequence having, in the amino acid sequence of SEQ ID NO: 2, (b) a substitution of leucine at position 111 with phenylalanine, or an amino acid sequence further having one or more amino acid substitutions selected from the following amino acid substitutions (a) to (g): (a) a substitution of isoleucine at position 105 with leucine; (c) a substitution of glutamic acid at position 124 with alanine; (d) a substitution of alanine at position 132 with glutamine or lysine; (e) a substitution of asparagine at position 140 with serine or methionine; (f) a substitution of serine at position 249 with aspartic acid; (g) a substitution of histidine at position 278 with tyrosine. [3] The L-pipecolic acid hydroxylase according to [1] to [2'], wherein the amino acid sequence of (1) or (1') is the amino acid sequence of SEQ ID NO: 2, further comprising substitutions of one or more amino acids at positions 5, 23, and 282 with other amino acids. [4] The L-pipecolic acid hydroxylase according to [3], wherein the amino acid sequence of (1) or (1') is the amino acid sequence of SEQ ID NO: 2, further comprising one or more amino acid substitutions selected from the following amino acid substitutions (p) to (r): (p) substitution of phenylalanine at position 5 with tyrosine; (q) substitution of cysteine ​​at position 23 with alanine; (r) substitution of aspartic acid at position 282 with glutamic acid.[5] The L-pipecolic acid hydroxylase according to any one of [1] to [4], wherein the amino acid sequence of (1) is selected from the group consisting of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, and 122. [6] A nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase according to any one of [1] to [5]. [7] The nucleic acid according to [6], wherein the nucleic acid comprises the nucleotide sequence shown in (4) or (5) below: (4) A nucleotide sequence having one to several nucleotide substitutions, deletions, and / or additions in the nucleotide sequence shown in SEQ ID NO: 1; (5) A nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1. [8] A method for producing hydroxy-L-pipecolic acid, comprising contacting the L-pipecolic acid hydroxylase according to any one of [1] to [5], a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell, with L-pipecolic acid to produce hydroxy-L-pipecolic acid. [9] A method for producing hydroxy-L-pipecolic acid according to [8], wherein the microorganism or cell is a microorganism or cell transformed with the nucleic acid according to [6] or [7].

[10] A recombinant vector comprising the nucleic acid according to [6] or [7].

[11] A transformant comprising the recombinant vector according to

[10] .

[0017] According to the present invention, by introducing a mutation into a hydroxylase, an L-pipecolic acid hydroxylase having improved L-pipecolic acid hydroxylating activity and high stability can be provided. Furthermore, a method for producing hydroxy-L-pipecolic acid from L-pipecolic acid with high productivity and at low cost on an industrial scale can be provided by utilizing such an L-pipecolic acid hydroxylase.

[0018] 1 is a diagram showing the relationship between the αKG concentration in a McPH hydroxylation reaction solution and the absorbance at a wavelength of 250 nm in Example 5 (2). 2 is a diagram showing the relationship between the amount of 5-hydroxy-L-pipecolic acid produced (conversion rate) and the degree of decrease in absorbance for wild-type McPH and McPH mutants in Example 5 (3). 3 is a diagram showing the relationship between the heat treatment temperature and the amount of 5-hydroxy-L-pipecolic acid produced for wild-type McPH and McPH mutants in Example 7.

[0019] 1. L-pipecolic acid hydroxylase of the present invention The L-pipecolic acid hydroxylase of the present invention comprises any one of the following amino acid sequences (1) to (3): (1) an amino acid sequence represented by SEQ ID NO: 2 in which one or more of the 105th, 111th, 124th, 132nd, 140th, 249th, and 278th amino acids are substituted with other amino acids; (2) an amino acid sequence in which the amino acids other than the substituted amino acids in the amino acid sequence of (1) are modified and which has 90% or more sequence identity with the amino acid sequence of (1); (3) an amino acid sequence in which the amino acids other than the substituted amino acids in the amino acid sequence of (1) have one to several amino acid substitutions, deletions, and / or additions.

[0020] The L-pipecolic acid hydroxylase of the present invention has the ability to add a hydroxyl group to the carbon atom at position 3, 4, and / or 5 of L-pipecolic acid (hereinafter, may be referred to as "L-pipecolic acid hydroxylating activity"). The L-pipecolic acid hydroxylase of the present invention is particularly excellent in the ability to add a hydroxyl group to the carbon atom at position 5 of L-pipecolic acid.

[0021] The L-pipecolic acid hydroxylase of the present invention has higher L-pipecolic acid hydroxylating activity and higher stability than the conventionally known L-pipecolic acid hydroxylases comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. By using the L-pipecolic acid hydroxylase of the present invention, hydroxy-L-pipecolic acid can be produced from L-pipecolic acid with high productivity and at low cost.

[0022] The L-pipecolic acid hydroxylase of the present invention comprises an amino acid sequence represented by SEQ ID NO: 2 in which one or more of the amino acids at positions 105, 111, 124, 132, 140, 249, and 278 have been substituted with other amino acids (hereinafter, this may be referred to as "amino acid sequence (1)").

[0023] The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of the WP_030487089 protein (McPH) derived from Micromonospora chokoriensis.

[0024] Preferably, the L-pipecolic acid hydroxylase of the present invention has the amino acid sequence (1) of SEQ ID NO: 2, which has one or more amino acid substitutions selected from the following amino acid substitutions (hereinafter sometimes referred to as "mutations") (a) to (g): (a) substitution of isoleucine at position 105 with leucine (hereinafter may be referred to as "I105L"); (b) substitution of leucine at position 111 with phenylalanine (hereinafter may be referred to as "L111F"); (c) substitution of glutamic acid at position 124 with alanine (hereinafter may be referred to as "E124A"); (d) substitution of alanine at position 132 with glutamine or lysine (hereinafter may be referred to as "A132Q" or "A132K"); (e) substitution of asparagine at position 140 with serine or methionine (hereinafter may be referred to as "N140S" or "N140M"); (f) substitution of serine at position 249 with aspartic acid (hereinafter may be referred to as "S249D"); (g) substitution of histidine at position 278 with tyrosine (hereinafter sometimes referred to as “H278Y”)

[0025] The L-pipecolic acid hydroxylase of the present invention may have at least one of the amino acid substitutions (a) to (g) in the amino acid sequence of SEQ ID NO: 2, but may have more than one of the amino acid substitutions (a) to (g).

[0026] Examples of amino acid sequences having the (a) substitution include the amino acid sequences of SEQ ID NOs: 16, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (b) substitution include the amino acid sequences of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (c) substitution include the amino acid sequences of SEQ ID NOs: 6, 14, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (d) substitution include the amino acid sequences of SEQ ID NOs: 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (e) substitution include the amino acid sequences of SEQ ID NOs: 120 or 122. Examples of amino acid sequences having the substitution (f) include the amino acid sequences of SEQ ID NOs: 18, 26, 28, 118, 120, and 122. Examples of amino acid sequences having the substitution (g) include the amino acid sequences of SEQ ID NOs: 20, 22, 24, 26, 28, 52, and 54.

[0027] In terms of effectiveness, the L-pipecolic acid hydroxylase of the present invention may have the amino acid sequence (1) of SEQ ID NO: 2 in which one or more of the amino acids at positions 5, 23, and 282 have been substituted with other amino acids.

[0028] Preferably, the L-pipecolic acid hydroxylase of the present invention has the amino acid sequence (1) of SEQ ID NO: 2 further having one or more amino acid substitutions selected from the following amino acid substitutions (p) to (r): (p) substitution of phenylalanine at position 5 with tyrosine (hereinafter may be referred to as "F5Y"); (q) substitution of cysteine ​​at position 23 with alanine (hereinafter may be referred to as "C23A"); (r) substitution of aspartic acid at position 282 with glutamic acid (hereinafter may be referred to as "D282E");

[0029] Examples of amino acid sequences having the (p) substitution include the amino acid sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (q) substitution include the amino acid sequences of SEQ ID NOs: 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122. Examples of amino acid sequences having the (r) substitution include the amino acid sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122.

[0030] In terms of effect, the L-pipecolic acid hydroxylase of the present invention may have the amino acid sequence (1) in which the amino acid at position 90 in the amino acid sequence represented by SEQ ID NO: 2 is further substituted with another amino acid.

[0031] Preferably, the amino acid sequence (1) of the L-pipecolic acid hydroxylase of the present invention is an amino acid sequence represented by SEQ ID NO: 2, further comprising the following amino acid substitution (s): (s) substitution of aspartic acid at position 90 with serine (hereinafter, sometimes referred to as "D90S").

[0032] Examples of amino acid sequences having the substitution (s) include the amino acid sequences of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 24, 28, and 30.

[0033] The relationship between the amino acid substitutions (a) to (g), (p) to (r), and (s) and the sequence numbers is shown in the table below.

[0034] Each of the amino acid substitutions (a) I105L, (b) L111F, (c) E124A, (d) A132Q or A132K, (e) N140S or N140M, (f) S249D, and (g) H278Y of the present invention can improve L-pipecolic acid hydroxylating activity.

[0035] As used herein, improvement in L-pipecolic acid hydroxylating activity means, for example, that the activity of L-pipecolic acid hydroxylase is improved by 10% or more, 20% or more, or 50% or more compared to before the introduction of the amino acid substitution. The L-pipecolic acid hydroxylating activity can be measured, for example, by the amount of product or the amount of substrate consumed, as described below.

[0036] For example, as shown in Example 3 below, the L-pipecolic acid hydroxylating activity can be further improved by having the amino acid substitutions (s) D90S and (b) L111F. The L-pipecolic acid hydroxylating activity can be further improved by having the amino acid substitutions (a) I105L, (c) E124A, (f) S249D, and / or (g) H278Y in addition to (s) D90S and (b) L111F.

[0037] Furthermore, as shown in Example 6 below, by having the amino acid substitutions (s) D90S, (a) I105L, (b) L111F, (d) S249D, and / or (g) H278Y in addition to (c) E124A, the L-pipecolic acid hydroxylating activity can be further improved compared to the case of (c) E124A alone.

[0038] Furthermore, as shown in Example 8 below, (d) each of the amino acid substitutions A132Q and A132K can improve the L-pipecolic acid hydroxylation activity.

[0039] Furthermore, each of the amino acid substitutions of the present invention, (a) I105L, (b) L111F, (c) E124A, (d) A132Q or A132K, (e) N140S or N140M, (f) S249D, and (g) H278Y, can improve stability.

[0040] As used herein, "improved stability" means that the stability of L-pipecolic acid hydroxylase is improved by 10% or more, 20% or more, or 50% or more, compared to that before the amino acid substitution was introduced. The stability can be evaluated, for example, by measuring the enzyme activity after treatment at 35°C for 1 hour.

[0041] For example, as shown in Example 7 below, the amino acid substitutions (a) I105L, (b) L111F, (c) E124A, and (g) H278Y can improve stability. In addition, the amino acid substitutions (p) F5Y, (q) C23A, and (r) D282E can also improve stability.

[0042] Furthermore, as shown in Example 9 below, each of the amino acid substitutions (d) A132Q or A132K and (f) S249D can improve stability.

[0043] Furthermore, as shown in Example 10 below, (e) each amino acid substitution of N140S or N140M can also improve stability.

[0044] As shown in Examples 3 and 6 below, the amino acid substitution of (s)D90S is thought to contribute to an improvement in the L-pipecolic acid hydroxylating activity, but as shown in Example 7 below, it is thought not to contribute to an improvement in stability.

[0045] As the L-pipecolic acid hydroxylase of the present invention, the amino acid sequence (1) is preferably one having the amino acid mutations (a) I105L, (b) L111F, and (c) E124A in the amino acid sequence shown in SEQ ID NO: 2, because it has excellent L-pipecolic acid hydroxylating activity and stability, and further preferably one having the amino acid mutations (a) I105L, (b) L111F, (c) E124A, (p) F5Y, (q) C23A, and (r) D282E, and particularly preferably one having the amino acid mutation (f) S249D in addition to the amino acid mutations (a) I105L, (b) L111F, (c) E124A, (p) F5Y, (q) C23A, and (r) D282E.

[0046] Furthermore, since those skilled in the art can predict that similar effects can be achieved, the L-pipecolic acid hydroxylase of the present invention includes an amino acid sequence in which amino acids other than the substituted amino acids in the amino acid sequence of (1) have been modified, the amino acid sequence having 90% or more sequence identity with the amino acid sequence of (1), and the amino acid sequence having L-pipecolic acid hydroxylating activity. From the viewpoint of effect, the amino acid sequence preferably has 95% or more, more preferably 98% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of (1).

[0047] For example, when the amino acid sequence of (1) is an amino acid sequence in which the 111th amino acid in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid (phenylalanine), the amino acid sequence includes an amino acid sequence in which an amino acid other than the phenylalanine in the amino acid sequence has been modified, which has 90% or more sequence identity with the amino acid sequence, and which has L-pipecolic acid hydroxylating activity.

[0048] Here, "modification" of an amino acid includes, for example, deletion, insertion, substitution and / or addition of an amino acid.

[0049] For example, the amino acid sequence may be one in which amino acids other than the substituted amino acids in the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122 have been modified, and which has an amino acid sequence that has 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122.

[0050] For example, the amino acid sequence may include an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 12, in which amino acids other than the 111th amino acid (phenylalanine), the 5th amino acid (tyrosine), the 23rd amino acid (alanine), and the 282nd amino acid (glutamic acid) have been modified, and which has L-pipecolic acid hydroxylating activity.

[0051] As used herein, sequence identity refers to the percentage of identical nucleotides or amino acids shared between two sequences when the two sequences are optimally aligned in a nucleotide sequence or amino acid sequence. That is, identity can be calculated as follows: identity = (number of identical 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, searches and analyses of nucleotide sequence or amino acid sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. Specific techniques for these analysis methods are well known to those skilled in the art.

[0052] Furthermore, since those skilled in the art can predict that a similar effect can be achieved, the L-pipecolic acid hydroxylase of the present invention includes an amino acid sequence in which one to several amino acids other than the substituted amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence of (1) above, and which has L-pipecolic acid hydroxylating activity.

[0053] For example, when the amino acid sequence of (1) is an amino acid sequence in which the 111th amino acid in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid (phenylalanine), the amino acid sequence may be an amino acid sequence in which one to several amino acids other than the phenylalanine are deleted, inserted, substituted, and / or added in the same amino acid sequence, and which contains an amino acid sequence having L-pipecolic acid hydroxylating activity.

[0054] For example, the amino acid sequence may be SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120, or 122, which is the amino acid sequence of (1) above in which one to several amino acids other than the substituted amino acid(s) are deleted, inserted, substituted, and / or added, and which contains an amino acid sequence having L-pipecolic acid hydroxylating activity.

[0055] For example, the amino acid sequence may include an amino acid sequence having L-pipecolic acid hydroxylating activity, which is an amino acid sequence represented by SEQ ID NO: 12 in which one or more amino acids other than the 111th amino acid (phenylalanine), the 5th amino acid (tyrosine), the 23rd amino acid (alanine), and the 282nd amino acid (glutamic acid) have been deleted, inserted, substituted, and / or added.

[0056] Here, "one to more than one" refers to any number that does not impair the performance of the L-pipecolic acid hydroxylase of the present invention, and is usually 1 to 100. From the viewpoint of effectiveness, the number is preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0057] In addition, in the case of substitution, conservative substitution, in which one amino acid is replaced with another amino acid that is biologically and / or chemically similar, is preferred, such as replacing one hydrophobic amino acid with another hydrophobic amino acid, one acidic amino acid with another acidic amino acid, or one basic amino acid with another basic amino acid.

[0058] In the present invention, the activity of L-pipecolic acid hydroxylase can be evaluated by the hydroxy-L-pipecolic acid-producing activity (U / g) or the αKG-consuming activity (U / g).

[0059] Hydroxy-L-pipecolic acid-producing activity can be determined by contacting the target enzyme with L-pipecolic acid and measuring the amount of hydroxy-L-pipecolic acid converted from L-pipecolic acid. For example, a reaction solution containing 0.00001 mol / L to 1 mol / L of L-pipecolic acid as a substrate, and 0.00001 mol / L to 1 mol / L of alpha-ketoglutaric acid (hereinafter sometimes referred to as "αKG"), 0 mol / L to 1 mol / L of L-ascorbic acid, and 0 mol / L to 0.1 mol / L of divalent iron ions as reaction aids is added to the reaction solution at an appropriate temperature (e.g., about 10°C to 45°C) and pressure (e.g., about atmospheric pressure), and a microorganism or cell capable of producing the target enzyme, a processed product of the microorganism or cell, or a protein purified from the microorganism or cell is added and contacted (reacted), and the amount of hydroxy-L-pipecolic acid produced is measured. This allows calculation of the hydroxy-L-pipecolic acid-producing activity (U / g) per cell mass (unit: g or turbidity) or total protein amount (unit: g) added to the reaction solution, where unit (U) represents the ability to produce 1 μmole of hydroxy-L-pipecolic acid per minute.

[0060] The αKG consuming activity (U / g) can be determined from the αKG concentration in the reaction mixture or the amount of αKG lost.

[0061] The present inventors have developed a method for estimating the αKG concentration and the amount of αKG loss by hydrazonizing αKG in a reaction solution and measuring the absorbance of the resulting hydrazone compound.

[0062] A specific method, for example, 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, which is reacted with αKG to produce a hydrazone compound, and the absorbance at a specific wavelength is measured. The resulting absorbance is compared with the absorbance of a control solution containing the hydrazone compound, thereby estimating the concentration and amount of reduction of the hydrazone compound in the reaction solution, i.e., the concentration and amount of reduction of αKG in the reaction solution.

[0063] If a calibration curve is prepared in advance by determining the relationship between the concentration and absorbance of αKG in the reaction mixture and the amount of αKG lost, the amount of αKG consumed in the reaction can be estimated from the measured absorbance using the calibration curve. The amount of αKG consumed is approximately equal to the amount of hydroxylated L-pipecolic acid produced, and can therefore be evaluated as the enzymatic activity of the hydroxylase.

[0064] Examples of controls that can be used include an αKG-containing reaction solution in which the reaction has not progressed, an αKG-containing reaction solution in which no enzyme has been added, an αKG-containing reaction solution in which inactivated hydroxylase has been added, an αKG-containing reaction solution in which recombinant cell lysate that does not express hydroxylase has been added, and an αKG-containing reaction solution in which the reaction has been stopped at reaction time 0.

[0065] The wavelength for measuring absorbance is preferably near the wavelength that gives the maximum extinction coefficient of the hydrazone compound produced, and can be appropriately selected depending on the hydrazine compound used. For example, when SCA is used, a wavelength of about 250 nm is preferred, and when DNPH is used, a wavelength of about 430 nm is preferred. It is also preferable to select a wavelength that is less affected by other components contained in the reaction solution.

[0066] The amount of hydrazine compound used is not particularly limited as long as it is an amount that can sufficiently react with αKG. It is usually at least 1 molar amount of αKG, preferably 2 to 10,000 molar amounts, and more preferably 5 to 1,000 molar amounts. The pH during the reaction of αKG with the hydrazine compound is usually in the range of 1 to 10, preferably 2 to 8, and more preferably 3 to 7. The reaction temperature is usually in the range of 0 to 100°C, preferably 10 to 80°C, and more preferably 20 to 60°C. The reaction time can be appropriately selected depending on the reaction temperature, and can be extended without any problems. However, the reaction time is usually 0 minutes (measured immediately after addition) to 24 hours. To obtain stable results, it is preferably 10 minutes to 2 hours, more preferably 30 minutes to 1 hour.

[0067] Since L-ascorbic acid absorbs light in the ultraviolet region and reacts with hydrazine compounds, it is not preferable to add high concentrations of L-ascorbic acid to the reaction solution. The concentration of L-ascorbic acid in the reaction solution is usually 10 molar equivalents or less of αKG, preferably 1 molar equivalent or less, more preferably 0.5 molar equivalent or less, and particularly preferably 0.1 molar equivalent or less.

[0068] According to the above method, the αKG concentration and the amount of αKG loss can be estimated by measuring absorbance, and the activity of L-pipecolic acid hydroxylase can be easily evaluated in large quantities (several hundred samples) at once.

[0069] In the present invention, the stability of L-pipecolic acid hydroxylase can be evaluated based on the residual activity after heat treatment. For example, the activity of the enzyme to be measured can be measured after maintaining the enzyme at a certain temperature for a certain period of time, and then compared with the activity of the enzyme without heat treatment.

[0070] The L-pipecolic acid hydroxylase of the present invention can be produced from the amino acid sequence represented by SEQ ID NO: 2 by methods known to those skilled in the art, such as site-directed mutagenesis and PCR.

[0071] The L-pipecolic acid hydroxylase of the present invention can also be produced by culturing a transformant containing a nucleic acid encoding it and isolating and purifying the L-pipecolic acid hydroxylase from the resulting culture. The nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., coding strand) or the antisense strand (i.e., non-coding strand).

[0072] Examples of DNA from which the DNA encoding the L-pipecolic acid hydroxylase of the present invention can be derived include DNA cloned from Micromonospora chokoriensis. For example, the DNA can be obtained by known PCR or hybridization from a DNA fraction prepared from cells or tissues derived from Micromonospora chokoriensis. Other examples include full-length L-pipecolic acid hydroxylase cDNA directly amplified by reverse transcriptase-PCR using total RNA or mRNA fractions prepared from cells or tissues derived from Micromonospora chokoriensis as a template. Such DNA can be converted (mutated) using a known kit, such as Mutan®-Super Express Km (TAKARA BIO INC.) or Mutan® K (TAKARA BIO INC.), according to a known method such as the ODA-LA PCR method, the gapped duplex method, or the Kunkel method, or a method similar thereto, to obtain a DNA encoding the L-pipecolic acid hydroxylase of the present invention. Alternatively, the DNA can be obtained by converting the cDNA cloned by colony or plaque hybridization or PCR from a cDNA library prepared by inserting the total RNA or mRNA fragment into an appropriate vector, and then converting the cDNA according to the above-mentioned method. The vector used for the library may be a bacteriophage, a plasmid, a cosmid, a phagemid, or the like.

[0073] Examples of nucleic acids encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention include nucleic acids containing the nucleotide sequences shown in (4) or (5) below: (4) A nucleotide sequence having one to several nucleotide substitutions, deletions, and / or additions in the nucleotide sequence shown in SEQ ID NO: 1. (5) A nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0074] The nucleotide sequence represented by SEQ ID NO: 1 is a synthetic nucleotide sequence obtained by optimizing the codons of the Micromonospora chokoriensis gene encoding the amino acid sequence of SEQ ID NO: 2 for expression in E. coli. Not only the Micromonospora chokoriensis gene, but also nucleic acids whose codons have been optimized in accordance with the host to be transformed are naturally encompassed in the nucleic acid encoding the protein having L-pipecolic acid hydroxylating activity of the present invention.

[0075] The nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention includes a nucleic acid containing a base sequence having one to several base substitutions, deletions, and / or additions in the base sequence represented by SEQ ID NO: 1. In the case of substitutions, conservative substitutions in which one base is replaced with another base that is biologically and / or chemically similar are preferred.

[0076] Here, "one to more than one" refers to any number that does not impair the performance of the nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention, and is usually 1 to 100. From the viewpoint of effectiveness, the number is preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0077] Furthermore, examples of nucleic acids encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention include those containing a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more sequence identity with the base sequence represented by SEQ ID NO: 1 in terms of effectiveness.

[0078] Furthermore, the nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention also includes a nucleic acid containing a nucleotide sequence that hybridizes under stringent conditions with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 1, as long as it encodes a polypeptide having L-pipecolic acid hydroxylating activity.

[0079] Here, "a base sequence that hybridizes under stringent conditions" means a base sequence of DNA obtained by using DNA as a probe under stringent conditions, using colony hybridization, plaque hybridization, Southern blot hybridization, or the like.

[0080] Examples of stringent conditions for colony hybridization or plaque hybridization include hybridization at 65° C. in the presence of 0.7 mol / L to 1 mol / L aqueous sodium chloride solution using a filter on which colony- or plaque-derived DNA or a fragment of said DNA is immobilized, followed by washing the filter with 0.1×SSC solution (1×SSC consists of a 150 mmol / L aqueous sodium chloride solution and a 15 mmol / L aqueous sodium citrate solution) at 65° C. The hybridization can be carried out according to the method described in, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

[0081] Furthermore, various nucleic acids resulting from codon degeneracy are also encompassed within the nucleic acid of the present invention, as long as they encode a polypeptide having L-pipecolic acid hydroxylating activity. Codon degeneracy refers to the phenomenon in which different codons (base sequences consisting of three nucleotides) encode the same amino acid during the translation process from nucleic acid to amino acid. For example, for alanine (A), 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 identical, the nucleic acid sequence encoding it can retain diversity within the range of codon degeneracy, resulting in the production of identical polypeptides. Thus, nucleic acid sequences encoding certain identical polypeptides are equivalent to each other. Methods for obtaining such equivalent nucleic acid sequences include site-directed mutagenesis, as described below, as well as modification of nucleic acid sequences using known genetic engineering techniques or the use of artificially synthesized nucleic acids.

[0082] Those skilled in the art can obtain a nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention by appropriately substituting, deleting, inserting, and / or adding base substitutions that result in desired mutations in the nucleic acid represented by SEQ ID NO: 1 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)) or the like.

[0083] Furthermore, those skilled in the art can use site-directed saturation mutagenesis (Nucleic Acids Research, Volume 32, Issue 14, July 15, 2004, Page e115) or the like to obtain a mutant library consisting of a collection of amino acids in which specific amino acid residues in the L-pipecolic acid hydroxylase of the present invention have been substituted with one or more other amino acids. Primers used in site-directed saturation mutagenesis can be purchased from, for example, Thermo Scientific or Twist Bioscience.

[0084] Examples of the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention and the nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase include, but are not limited to, the sequences listed in Table 2 below, which are also described in the Examples below.

[0085]

[0086] In the method for producing hydroxy-L-pipecolic acid of the present invention described below, the L-pipecolic acid hydroxylase may be directly used in the reaction of L-pipecolic acid as a substrate. However, 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 solution containing the enzyme obtained by culturing the microorganism or cell.

[0087] The microorganisms or cells capable of producing the L-pipecolic acid hydroxylase of the present invention may be microorganisms or cells that originally have the ability to produce the L-pipecolic acid hydroxylase, or microorganisms or cells to which the production ability has been imparted by breeding. The microorganisms or cells may be live or dead, and for example, resting cells may be suitably used. Examples of types of microorganisms or cells capable of producing the L-pipecolic acid hydroxylase of the present invention include those described below as "host microorganisms" or "host cells."

[0088] As a means for imparting the above-mentioned productivity by breeding, known methods such as genetic recombination (transformation), mutation, etc. Examples of transformation methods include a method of introducing a target DNA and a method of enhancing expression of a target DNA by modifying an expression regulatory sequence such as a promoter on a chromosome.

[0089] Among these, it is preferable to use a microorganism or cell transformed with a nucleic acid (DNA) encoding the amino acid sequence of the L-pipecolic acid hydroxylase of the present invention.

[0090] As described above, the DNA encoding the L-pipecolic acid hydroxylase of the present invention can be obtained by cloning and converting the DNA by PCR using chromosomal DNA derived from Micromonospora chokoriensis as a template and appropriate primers.

[0091] Furthermore, the DNA encoding the L-pipecolic acid hydroxylase of the present invention can be obtained by preparing a full-length L-pipecolic acid hydroxylase cDNA by directly amplifying the cDNA by RT-PCR using total RNA or mRNA derived from Micromonospora chokoriensis as a template, as described above, and then cloning and converting the cDNA by PCR using appropriate primers.

[0092] For example, the gene expression vector of the present invention is provided by inserting the DNA encoding the L-pipecolic acid hydroxylase of the present invention obtained as described above into a known expression vector in an expressible configuration. Then, by transforming a host cell with the expression vector, a transformant into which the DNA encoding the protein of the present invention has been introduced can be obtained. Transformants can also be obtained by expressibly incorporating the DNA encoding the protein of the present invention into the chromosomal DNA of the host by a technique such as homologous recombination.

[0093] As used herein, the term "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 and introducing the vector into the host organism. Examples of such vectors include, but are not limited to, plasmids, viruses, phages, and cosmids. Preferably, the expression vector is a plasmid.

[0094] As used herein, the term "transformant" refers to a microorganism or cell into which a gene of interest has been introduced using an expression vector or the like, and which is now capable of expressing a desired trait associated with a protein having a desired function.

[0095] Specific examples of methods for producing transformants include a method in which DNA encoding the L-pipecolic acid hydroxylase of the present invention is introduced into a plasmid vector, phage vector, or viral vector that stably exists in a host cell, and the constructed expression vector is then introduced into the host cell, or a method in which the DNA is directly introduced into the host genome and the genetic information is transcribed and translated. In this case, it is preferable to link a suitable promoter upstream of the 5'-end of the DNA in the host, and it is even more preferable to link a terminator downstream of the 3'-end. 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. For example, vectors, promoters, and terminators described in detail in "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan" can be used.

[0096] The host microorganism to be transformed to express the L-pipecolic acid hydroxylase of the present invention is not particularly limited, as long as the host itself does not adversely affect the substrate L-pipecolic acid or the target product hydroxy-L-pipecolic acid. Examples of the host microorganism include the following microorganisms:

[0097] Bacteria for which host-vector systems have been established, such as bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, and Lactobacillus.

[0098] Actinomycetes belonging to the genus Rhodococcus, Streptomyces, etc., for which host-vector systems have been established.

[0099] Yeasts for which host-vector systems have been established, such as those belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, and Candida.

[0100] Fungi for which host-vector systems have been established, such as those belonging to the genera Neurospora, Aspergillus, Cephalosporium, and Trichoderma.

[0101] The procedures for preparing a transformant, the construction of a recombinant vector suitable for the host, and the method for culturing the host can be carried out in accordance with techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (e.g., the method described in Green et al., Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)).

[0102] Specific examples of preferred host microorganisms, preferred transformation techniques for each microorganism, vectors, promoters, terminators, etc. are given below, but the present invention is not limited to these examples.

[0103] In the genus Escherichia, particularly Escherichia coli, examples of plasmid vectors include pBR and pUC-based plasmids, and examples of promoters include those derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac and trp fusion), λ phage PL, PR, T7 phage, etc. Examples of terminators include those derived from trpA, phage, and rrnB ribosomal RNA.

[0104] In the genus Bacillus, examples of vectors include pUB110-based plasmids and pC194-based plasmids, and they can also be integrated into chromosomes. As promoters and terminators, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, and α-amylase can be used.

[0105] For the genus Pseudomonas, examples of vectors include general host vector systems established for Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the decomposition of toluene compounds, and broad-host-range vectors based on the TOL plasmid (containing genes necessary for autonomous replication derived from RSF1010, etc.) pKT240 (Gene, 26, 273-82 (1983)).

[0106] For the genus Brevibacterium, particularly Brevibacterium lactofermentum, examples of vectors include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). As promoters and terminators, various promoters and terminators used in Escherichia coli can be used.

[0107] For the genus Corynebacterium, particularly Corynebacterium glutamicum, examples of vectors include plasmid vectors such as pCS11 (Japanese Patent Laid-Open Publication No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)).

[0108] For the genus Saccharomyces, particularly Saccharomyces cerevisiae, vectors include YRp, YEp, YCp, and YIp plasmids. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase can be used.

[0109] In the genus Schizosaccharomyces, examples of vectors include the plasmid vector derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Bio Inc. and can be easily used.

[0110] Among the Aspergillus genus, Aspergillus niger, Aspergillus oryzae, and the like have been most extensively studied among fungi, and integration into plasmids or chromosomes is available, and promoters derived from extracellular proteases or amylases are available (Trends in Biotechnology 7, 283-287 (1989)).

[0111] In addition to the above, host-vector systems have been established for various microorganisms, and these can be used as appropriate.

[0112] In addition to microorganisms, various host-vector systems have been established in plants and animals. In particular, systems for expressing large amounts of heterologous proteins in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)) and plants such as rapeseed, corn, and potato, as well as systems using cell-free protein synthesis systems such as Escherichia coli cell-free extracts and wheat germ, have been established and can be suitably used.

[0113] Examples of processed products of microorganisms or cells capable of producing the L-pipecolic acid hydroxylase of the present invention include cell preparations such as those obtained by treating the microorganisms or cells with organic solvents such as acetone, dimethyl sulfoxide, toluene, etc., or surfactants, or by freeze-drying, or by physically or enzymatically disrupting the microorganisms or cells; purified enzymes (including partially purified enzymes) obtained by extracting enzyme fractions from microorganisms or cells as crude or purified products; and those obtained by immobilizing these on carriers such as polyacrylamide gel and carrageenan gel.

[0114] Examples of a culture medium containing the L-pipecolic acid hydroxylase of the present invention obtained by culturing a microorganism or cells capable of producing the enzyme include a suspension of the microorganism or cells and a liquid medium; and, when the microorganism or cells are secretory expression cells, a supernatant obtained by removing the microorganism or cells by centrifugation or a concentrate thereof. The culture may be carried out under any conditions suitable for culturing the microorganism or cells, and may also be under conditions appropriately adjusted to optimize the activity, physical properties, productivity, etc. of the L-pipecolic acid hydroxylase of the present invention. When Escherichia coli is used as a host, the culture temperature is typically 20°C to 50°C, preferably 20°C to 42°C, and more preferably 28°C to 38°C. The pH of the culture medium during culture is typically 3 to 9, preferably 5 to 8, and more preferably 6 to 7.5. The pH of the culture medium during culture can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. The culture time is typically 3 hours to 7 days, preferably 4 hours to 5 days, and more preferably 5 hours to 3 days. These conditions are preferably selected appropriately depending on the microorganism or cell used.

[0115] 2. Production Method of Hydroxy-L-pipecolic Acid of the Present Invention The production method of hydroxy-L-pipecolic acid of the present invention comprises producing hydroxy-L-pipecolic acid by contacting L-pipecolic acid with the L-pipecolic acid hydroxylase of the present invention, a microorganism or cell capable of producing the enzyme, a treated product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell (hereinafter, these may be referred to as "the L-pipecolic acid hydroxylase of the present invention, etc.").

[0116] Examples of hydroxy-L-pipecolic acid include (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, and (5R)-hydroxy-L-pipecolic acid. From the viewpoint of efficacy, (5S)-hydroxy-L-pipecolic acid or (5R)-hydroxy-L-pipecolic acid is preferred, and (5S)-hydroxy-L-pipecolic acid is particularly preferred.

[0117] The production method of the present invention can use a purified or roughly purified L-pipecolic acid hydroxylase of the present invention, a microorganism or cell capable of producing the L-pipecolic acid hydroxylase of the present invention (e.g., a transformant having DNA encoding the L-pipecolic acid hydroxylase of the present invention), a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell. Among these, it is preferable to use a microorganism or cell capable of producing the L-pipecolic acid hydroxylase of the present invention, a processed product of the microorganism or cell, and / or a culture solution containing the enzyme obtained by culturing the microorganism or cell (e.g., a transformant having DNA encoding the L-pipecolic acid hydroxylase of the present invention), and it is more preferable to use a transformant having DNA encoding the L-pipecolic acid hydroxylase of the present invention.

[0118] In the production method of the present invention, multiple types of L-pipecolic acid hydroxylases may be used in combination.

[0119] The amount of the L-pipecolic acid hydroxylase or the like of the present invention to be contacted with L-pipecolic acid is not particularly limited, as long as it is an amount that can produce hydroxy-L-pipecolic acid. For example, when the microorganism or cells are added to a reaction solution containing L-pipecolic acid, they are added so that the concentration of the microorganism or cells in the reaction solution is typically about 0.1 w / v% to 50 w / v%, preferably 1 w / v% to 20 w / v%, based on wet cell weight. When the treated product or culture solution is added to a reaction solution containing L-pipecolic acid, the specific activity of the L-pipecolic acid hydroxylase used is determined, and an amount is added so that the cell concentration in the reaction solution reaches the above-mentioned cell concentration. Here, w / v% means weight / volume %.

[0120] The method of contact is not particularly limited, and for example, L-pipecolic acid as a substrate can be added to a liquid containing the L-pipecolic acid hydroxylase of the present invention, etc. Alternatively, the L-pipecolic acid hydroxylase of the present invention, etc. may be added to a liquid containing L-pipecolic acid as a substrate. When L-pipecolic acid comes into contact with the L-pipecolic acid hydroxylase of the present invention, etc., L-pipecolic acid is hydroxylated to produce hydroxy-L-pipecolic acid.

[0121] The amount of L-pipecolic acid can be appropriately selected depending on the amount of hydroxy-L-pipecolic acid to be produced. L-pipecolic acid can be used so that the substrate concentration in a liquid containing L-pipecolic acid, the L-pipecolic acid hydroxylase of the present invention, and the like (hereinafter sometimes referred to as a "reaction liquid") is usually 0.01 w / v % to 90 w / v %, preferably 0.1 w / v % to 30 w / v %.

[0122] L-pipecolic acid, the L-pipecolic acid hydroxylase of the present invention, and the like may be added all at once at the beginning, or may be added continuously or intermittently from the viewpoint of reducing the influence of substrate inhibition of the enzyme or increasing the accumulated concentration of the product.

[0123] The contact is preferably carried out in the presence of αKG and divalent iron ions.

[0124] αKG is usually added in an equimolar amount or more, preferably equimolar to 2-fold molar amounts, relative to the substrate L-pipecolic acid. αKG may be added all at once initially, or may be added continuously or intermittently from the viewpoint of reducing the influence of any inhibitory effects on the enzyme or increasing the accumulated concentration of the product. Alternatively, an inexpensive compound that can be metabolized by the host, such as glucose, may be added instead of αKG and allowed to metabolize by the host, with the αKG produced during the process being used in the reaction.

[0125] Divalent iron ions are preferably used at a concentration in the reaction solution of typically 0.00001 mol / L to 0.1 mol / L, preferably 0.0001 mol / L to 0.01 mol / L. Divalent iron ions can be added all at once initially as iron sulfate or the like. Furthermore, if the divalent iron ions added during the reaction are oxidized to trivalent iron or precipitates form and are reduced, it is also effective to add additional iron. Note that if the L-pipecolic acid hydroxylase or the like of the present invention already contains a sufficient amount of divalent iron ions, it is not necessarily necessary to add them.

[0126] The contact is preferably carried out in the presence of L-ascorbic acid. The concentration of L-ascorbic acid in the reaction solution is usually 0.00001 mol / L to 0.05 mol / L, preferably 0.0001 mol / L to 0.02 mol / L, and particularly preferably 0.0001 mol / L to 0.1 mol / L. The addition of L-ascorbic acid can reduce the oxidation of divalent iron ions.

[0127] The contact can usually be carried out in an aqueous medium or a mixture of an aqueous medium and an organic solvent, but from the viewpoint of reducing post-treatment and industrial load, it is preferably carried out in an aqueous medium.

[0128] Examples of aqueous media include water and known buffers such as Good's buffer, phosphate buffer, Tris buffer, and borate buffer. The organic solvent used is preferably one that has high solubility for the substrate L-pipecolic acid, and examples of such organic solvents that can be used include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, and dimethyl sulfoxide. Furthermore, the organic solvent may be one that can efficiently remove reaction by-products, and examples of such organic solvents that can be used include ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane.

[0129] The contacting can be carried out, for example, under a pressure of about atmospheric pressure. The contacting temperature is not particularly limited as long as it is a temperature at which L-pipecolic acid is hydroxylated to produce hydroxy-L-pipecolic acid, but is usually 4°C to 60°C, preferably 10°C to 45°C, and particularly preferably 15°C to 40°C. The contacting can also be carried out under conditions of usually pH 3 to 11, preferably pH 5 to 8. The contacting time is not particularly limited as long as it is a time at which L-pipecolic acid is hydroxylated to produce hydroxy-L-pipecolic acid, but is usually 10 minutes or more, preferably 30 minutes or more, and is usually 90 hours or less, preferably 72 hours or less.

[0130] The produced hydroxy-L-pipecolic acid can be purified by separating the bacterial cells, proteins, and the like in the reaction solution by a separation or purification method known to those skilled in the art, such as centrifugation or membrane treatment, and then by an appropriate combination of extraction with an organic solvent such as 1-butanol or tert-butanol, distillation, column chromatography using an ion exchange resin or silica gel, crystallization at the isoelectric point, crystallization with monohydrochloride, dihydrochloride, calcium salt, and the like, and the like.

[0131] According to the production method of the present invention, hydroxy-L-pipecolic acid, which is useful as an intermediate for pharmaceuticals, can be produced industrially at high productivity and low cost.

[0132] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0133] In the Examples, amino acids may be represented by the following abbreviations.

[0134]

[0135] In the Examples, for example, the notation "F5Y" means that the fifth phenylalanine in the amino acid sequence represented by SEQ ID NO:2 is substituted with tyrosine.

[0136] In the examples, unless otherwise specified, "%" means "weight / volume %".

[0137] Example 1: Preparation of McPH mutants and expression plasmids containing the same Using a method similar to that described in the Examples of Patent Document 4 (WO 2017 / 057730), a plasmid was prepared containing a McPH gene (SEQ ID NO: 3) in which three mutations, F5Y, C23A, and D282E, had been introduced into the wild-type McPH gene (SEQ ID NO: 1) encoded by the pJ411 plasmid. The McPH mutant with the three mutations introduced was named McPH-m3a (SEQ ID NO: 4), and the plasmid was named pJ411-McPH-m3a. Here, McPH-m3a is the same as the triple mutant McPHm11 in Patent Document 4.

[0138] Using pJ411-McPH-m3a as a template, PCR was performed according to a standard method using primers of SEQ ID NO: 55 and SEQ ID NO: 56, and PrimeSTAR (registered trademark) Max DNA Polymerase (manufactured by Takara Bio Inc.) to obtain a DNA fragment containing the McPH-m3a gene.

[0139] On the other hand, the pKW32 plasmid was cleaved with the restriction enzymes MfeI and XbaI, subjected to 1% agarose gel electrophoresis, and extracted from the agarose gel using a GEL / PCR Purification Mini Kit (manufactured by Chiyoda Science Co., Ltd.) according to the attached protocol, and the cleaved fragment was purified. Here, the pKW32 plasmid is the plasmid described in Reference Example 1 of Japanese Patent No. 5613660.

[0140] The two DNA fragments obtained above were ligated using the In-Fusion® HD Cloning Kit (manufactured by Takara Bio Inc.) according to the attached protocol, and the resulting DNA solution was used to transform Escherichia coli JM109 (manufactured by Takara Bio Inc.) according to the attached protocol. Furthermore, a miniprep method was performed using the QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) according to the attached protocol to prepare a plasmid. The nucleotide sequence of the obtained plasmid was confirmed and named pKW32-McPH-m3a.

[0141] Using the resulting pKW32-McPH-m3a as a template, PCR was performed according to standard methods using primers of SEQ ID NO: 57 and SEQ ID NO: 58, and PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.), to amplify a DNA fragment. The resulting amplified solution was treated with the restriction enzyme DpnI at 37°C for 1.5 hours to cleave the template plasmid. The resulting DNA solution was used to transform Escherichia coli JM109 (Takara Bio Inc.) in the same manner as above, and a miniprep method was performed in the same manner as above to prepare a plasmid. The nucleotide sequence of the resulting plasmid was confirmed, and it was named pKW32-McPH-m4a.

[0142] Furthermore, the plasmids shown in Table 4 were prepared in the same manner as above using the templates and primer sets shown in Table 4.

[0143]

[0144] Furthermore, the McPH gene was inserted into the pJ411 vector in the same manner as in the Examples of Patent Document 4 (WO2017 / 057730), and the resulting plasmid was designated pJ411-McPH. Furthermore, using the resulting pJ411-McPH plasmid as a template, the McPH gene was inserted into the pKW32 vector in the same manner as above, using primers of SEQ ID NO: 55 and SEQ ID NO: 56, and the resulting plasmid was designated pKW32-McPH.

[0145] Using the resulting pKW32-McPH as a template, PCR was carried out in the same manner as above using primers of SEQ ID NO: 71 and SEQ ID NO: 72 according to standard methods to obtain a DNA fragment containing the McPH gene.

[0146] On the other hand, PCR was carried out in the same manner as above using the pET24a vector as a template and primers of SEQ ID NO: 73 and SEQ ID NO: 74 according to a standard method to obtain a DNA fragment containing the pET24a vector.

[0147] The two resulting DNA fragments, a DNA fragment containing the McPH gene and a DNA fragment containing the pET24a vector, were ligated using an In-Fusion® HD Cloning Kit (Takara Bio Inc.) in the same manner as described above. The resulting DNA solution was used to transform Escherichia coli JM109 (Takara Bio Inc.) in the same manner as described above, and a miniprep method was performed in the same manner as described above to prepare a plasmid. The nucleotide sequence of the resulting plasmid was confirmed, and it was named pET-McPH.

[0148] Furthermore, using the template and primer set shown in Table 5, DNA fragments containing the McPH gene were obtained in the same manner as above, and each was ligated to a DNA fragment containing the pET24a vector. The resulting DNA solution was used to prepare the plasmids shown in Table 5 in the same manner as above.

[0149]

[0150] Example 2 Preparation of Culture Supernatant Using McPH Mutants Escherichia coli JM109 (Takara Bio Inc.) was transformed with each of the plasmids pKW32-McPH-m3a, pKW32-McPH-m4a, pKW32-McPH-m3a-E124A, pKW32-McPH-m5c, pKW32-McPH-m5c-E124A, pKW32-McPH-m5c-I105L, pKW32-McPH-m5c-S249D, and pKW32-McPH-m6a obtained in Example 1 according to the attached protocol, and the transformed strains were inoculated into LB liquid medium containing kanamycin as an antibiotic and cultured overnight at 37°C with shaking. The resulting culture was inoculated into LB medium containing 25 mg / L kanamycin, 0.4% by volume glycerol, and 0.2 mmol / L isopropyl-β-thiogalactopyranoside (IPTG), and cultured at 32°C for approximately 20 hours to induce protein expression. The cells were harvested from the culture and suspended in 100 mmol / L 2-morpholinoethanesulfonic acid (MES) buffer (pH 7.0). The container containing the suspension was immersed in ice water and subjected to ultrasonic disruption, after which the resulting cell lysate was centrifuged to obtain the supernatant. The resulting supernatant was used as the enzyme solution for measuring the activity of the McPH mutants.

[0151] Example 3: Measurement of activity of McPH mutants The activity of each enzyme solution obtained in Example 2 was measured as follows.

[0152] In a 2.0 mL plastic tube, 20 mmol / L of L-pipecolic acid, 20 mmol / L of αKG, 25 mmol / L of L-ascorbic acid, and 0.5 mmol / L of ferrous sulfate (FeSO ４ ), 2 mmol / L sodium citrate, 100 mmol / L MES buffer (pH 7.0), and 0.1 mL of a reaction solution prepared so that the enzyme solution obtained in Example 2 had a protein concentration of approximately 2 mg / mL was shaken at 15°C for 30 minutes. Then, 0.05 mL of 1 mol / L hydrochloric acid was added to each reaction solution to stop the reaction, followed by addition of 0.05 mL of 1 mol / L sodium hydroxide (NaOH) and 2 mmol / L copper sulfate (CuSO ４ ) was added, and the mixture was centrifuged to obtain the supernatant.

[0153] The resulting supernatant was analyzed by High Performance Liquid Chromatography (HPLC) to measure the concentration of hydroxy-L-pipecolic acid contained in the supernatant. The HPLC analysis conditions are shown in Table 6.

[0154]

[0155] The protein concentration in the resulting supernatant was measured using Quick Start Bradford 1x Dye Reagent (Bio-Rad) according to the attached protocol.

[0156] The L-pipecolic acid hydroxylating activity of each enzyme solution obtained in Example 2 was evaluated based on the concentration of hydroxy-L-pipecolic acid and the concentration of total protein contained in the supernatant obtained above. Specifically, the L-pipecolic acid hydroxylating activity (hydroxy-L-pipecolic acid-producing activity) was evaluated in units per total protein amount contained in the supernatant (U / g-protein). Here, unit (U) represents the ability to produce 1 μmole of hydroxy-L-pipecolic acid per minute. The results are shown in Table 7.

[0157]

[0158] As is clear from Table 7, the McPH mutants McPH-m3a-E124A, McPH-m4a, McPH-m5c, McPH-m5c-E124A, McPH-m5c-I105L, McPH-m5c-S249D, and McPH-m6a had improved L-pipecolic acid hydroxylation activity compared to the control McPH-m3a.

[0159] Specifically, McPH-m3a-E124A having the E124A mutation or McPH-m4a having the D90S mutation had L-pipecolic acid hydroxylation activity that was more than twice that of the control. McPH-m5c having the D90S and L111F mutations had L-pipecolic acid hydroxylation activity that was approximately three times that of the control. The strains carrying the S249D mutation (McPH-m5c-S249D) and the I105L mutation (McPH-m5c-I105L) in addition to the D90S and L111F mutations had higher L-pipecolic acid hydroxylation activity than McPH-m5c (D90S and L111F mutations). The strains carrying the E124A mutation (McPH-m5c-E124A) and the H278Y mutation (McPH-m6a) in addition to the D90S and L111F mutations had even higher L-pipecolic acid hydroxylation activity.

[0160] From this, it is considered that the D90S, I105L, L111F, E124A, S249D, and H278Y mutations each have the effect of improving L-pipecolic acid hydroxylation activity. Furthermore, when the D90S and L111F mutations are present, the effect of further improving L-pipecolic acid hydroxylation activity is achieved, and when the S249D, I105L, E124A, and / or H278 mutations are present in addition to the D90S and L111F mutations, the effect of further improving L-pipecolic acid hydroxylation activity is achieved.

[0161] The hydroxy-L-pipecolic acid produced in this example was cis-5-hydroxy-L-pipecolic acid.

[0162] Example 4 (1) Preparation of pET Plasmid-Type Mutant Library Using pET-McPH-m4a prepared in Example 1 as a template, PCR was performed according to a standard method using primers of SEQ ID NO: 75 and SEQ ID NO: 76, and PrimeSTAR (registered trademark) Max DNA Polymerase (manufactured by Takara Bio Inc.), to obtain DNA fragment No. 1.

[0163] Furthermore, PCR was carried out in the same manner as above using the template and primer set shown in Table 8 to obtain DNA fragments No. 2 to 5.

[0164] All of the obtained DNA fragments No. 1 to 5 were ligated using Gibson Assembly Master Mix (New England Biolabs) according to the attached protocol, and the resulting DNA solution was used to transform Escherichia coli DH5α (Takara Bio) in the same manner as described above. The transformed cells were then inoculated into LB liquid medium containing kanamycin as an antibiotic. Plasmids were prepared from the resulting bacteria by the miniprep method in the same manner as described above, and a pET plasmid mutant library (pET-McPH-cmb-lib) was created (Table 9).

[0165] (2) Preparation of pKW Plasmid-Type Mutant Library A pKW plasmid-type mutant library (pKW-McPH-cmb-lib) was prepared in the same manner as in Example 4(1), except that pKW32-McPH-m5c was used instead of pET-McPH-m4a as the template (Table 9).

[0166]

[0167]

[0168] In Table 9, for example, the notation "D / S" at amino acid residue 90 indicates that the 90th amino acid residue is either aspartic acid (D) or serine (S). The constructed mutant libraries pET-McPH-cmb-lib and pKW-McPH-cmb-lib are mutant libraries in which the 90th amino acid of the m3a mutant is changed to aspartic acid or serine, the 105th amino acid to isoleucine or leucine, the 111th amino acid to leucine or phenylalanine, the 124th amino acid to glutamic acid or alanine, the 249th amino acid to serine or aspartic acid, and the 278th amino acid to histidine or tyrosine, respectively.

[0169] Example 5 Evaluation of L-pipecolic acid hydroxylation activity by absorbance measurement (1) Preparation of bacterial cell lysate containing McPH or McPH mutant Escherichia coli BL21(DE3) (Novagen) was transformed using each of the plasmids pET-McPH, pET-McPH-m3a, pET-McPH-m4a, pET-McPH-m3b, and pET-McPH-m4b obtained in Example 1 according to the attached protocol, and the transformed cells were inoculated into an LB liquid medium containing kanamycin as an antibiotic and cultured overnight at 37°C with shaking.

[0170] The resulting culture solution was cultured in ZYM-5052 medium (10 g / L tryptone, 5 g / L yeast extract, 2 mmol / L magnesium sulfate (MgSO )), an autoinduction medium containing kanamycin. ４ ), 25 mmol / L disodium hydrogen phosphate (Na ２ HPO ４ ), 25 mmol / L potassium dihydrogen phosphate (KH ２ P.O. ４ ), 50 mmol / L ammonium chloride (NH ４ Cl), 5 mmol / L sodium sulfate (Na ２ SO ４ ), 0.5% glycerol, 0.05% glucose and 0.2% α-lactose) and cultured at 30°C for 20 hours to induce protein expression.

[0171] The cells were collected from the culture medium and frozen at −20° C. The frozen cells were left to stand at room temperature to thaw, and then lysed in a cell lysis buffer (100 mmol / L MES buffer (pH 7.0), 2 mmol / L MgSO ). ４The cells were suspended in a lysozyme solution (50 mmol / L MES buffer (pH 7.0), 100 mmol / L NaCl, 10 mg / mL lysozyme, 1 mg / mL DNase I (Worthington Biochemical), and 50% glycerol) containing 0.1% Triton (registered trademark) X-100 (Sigma-Aldrich) and 1 / 50th volume (volume ratio), and lysed by shaking at 20°C for 10 minutes. The resulting cell lysate was centrifuged to obtain cell lysate supernatants containing McPH and McPH mutants (McPH-m3a, McPH-m4a, McPH-m3b, or McPH-m4b).

[0172] (2) Relationship between αKG concentration and absorbance in McPH hydroxylation reaction solution 10 mmol / L L-pipecolic acid, αKG (appropriate concentration of 0 to 10 mmol / L), 1 mmol / L L-ascorbic acid, 10 mmol / L DTT, 0.5 mmol / L FeSO ４ To 18 μL of a reaction 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, and 2 μL of the bacterial cell lysate supernatant containing each McPH or McPH mutant prepared in Example 5(1) was added. The mixture was allowed to stand at 37°C for 30 minutes, and then the absorbance at a wavelength of 250 nm was measured using a microplate reader (Molecular Bio).

[0173] The relationship between the αKG concentration (in the range of 0 to 10 mmol / L) in the McPH hydroxylation reaction solution and the absorbance at a wavelength of 250 nm is shown in FIG.

[0174] As is clear from Figure 1, a linear correlation was observed between the αKG concentration in the McPH hydroxylation reaction solution and the absorbance at 250 nm, suggesting that the αKG concentration can be estimated by measuring the absorbance at 250 nm of the McPH hydroxylation reaction solution containing SCA.

[0175] (3) Relationship between the amount of hydroxy-L-pipecolic acid produced and absorbance 10 mmol / L L-pipecolic acid, 10 mmol / L αKG, 1 mmol / L ascorbic acid, 10 mmol / L DTT, 0.5 mmol / L FeSO ４ To 90 μL of a mixture containing 2 mmol / L sodium citrate and 100 mmol / L MES buffer (pH 7.0), 10 μL of the supernatant of the cell lysate containing each McPH or McPH variant (m3a, m4a, m3b, or m4b) prepared in Example 5(1) was added to initiate the hydroxylation reaction. The reaction was carried out at 20°C for 1 to 4 hours.

[0176] 20 μL of the resulting reaction solution was mixed with 100 μL of SCA solution, and after standing at 37°C for 30 minutes, the absorbance at a wavelength of 250 nm was measured using a microplate reader (Molecular Bio). The SCA solution was mixed in the same manner as above, and the absorbance at a wavelength of 250 nm was measured over the reaction time period from 0 to 4 hours. The decrease in absorbance was calculated as follows:

[0177] Decrease in absorbance = absorbance at any time between 1 and 4 hours of reaction time - absorbance at 0 hours of reaction

[0178] To confirm the production of 5-hydroxy-L-pipecolic acid, 40 μL of the reaction mixture obtained above was mixed with 20 μL of 1 mol / L hydrochloric acid, and then 20 μL of 1 mol / L NaOH and 2 mmol / L CuSO were added. ４ After adding 80 μL, the mixture was centrifuged and the resulting supernatant was subjected to HPLC analysis under the same conditions as in Example 3.

[0179] The relationship between the amount of 5-hydroxy-L-pipecolic acid produced (conversion rate) and the degree of decrease in absorbance for wild-type McPH and the McPH mutants is shown in FIG.

[0180] As is clear from FIG. 2, the greater the amount of 5-hydroxy-L-pipecolic acid produced, the greater the decrease in the absorbance, and there was a correlation of 0.94 between the amount of 5-hydroxy-L-pipecolic acid produced and the decrease in absorbance at a wavelength of 250 nm.

[0181] This demonstrates that the L-pipecolic acid hydroxylation activity of McPH and McPH mutants can be easily measured by measuring the decrease in absorbance at a wavelength of 250 nm of the McPH hydroxylation reaction solution containing SCA.

[0182]

[0183] Example 6: Preparation of transformants expressing pET plasmid-type mutant library or pKW plasmid-type mutant library and evaluation of L-pipecolic acid hydroxylation activity (1) Preparation of transformants expressing pET plasmid-type mutant library and evaluation of L-pipecolic acid hydroxylation activity Using the pET plasmid-type mutant library (pET-McPH-cmb-lib) prepared in Example 4 and pET-McPH-m3a-E124A prepared in Example 1, Escherichia coli BL21 (DE3) (Novagen) was transformed according to the attached protocol, and the transformed Escherichia coli was inoculated onto an LB agar medium containing kanamycin as an antibiotic and cultured at 23°C for 3 days to obtain colonies of transformed Escherichia coli on the LB agar medium.

[0184] 200 μL of LB liquid medium containing 25 mg / L kanamycin as an antibiotic was dispensed into a 96-well deep well plate (manufactured by Whatman), and the colonies obtained by the above culture were inoculated one by one and cultured with shaking at 30°C as a preculture.

[0185] Next, 200 μL of ZYM-5052 medium containing 25 mg / L kanamycin was dispensed into another 96-well deep well plate (Whatman), and 5 μL of each culture obtained in the preculture was inoculated and cultured with shaking at 37 ° C for 4 hours, followed by further culture with shaking at 20 ° C for 16 hours to induce protein expression. The resulting culture was centrifuged, the supernatant discarded, and the 96-well deep well plate containing the residue was frozen and stored at -80 ° C. The frozen 96-well deep well plate was left to stand at room temperature to thaw the bacterial cells, and 200 μL of bacterial cell lysis buffer was added and the plate was shaken at 20 ° C for 10 minutes to lyse the bacterial cells. The obtained bacterial cell lysate was centrifuged, and the bacterial cell lysate supernatant was obtained.

[0186] The L-pipecolic acid hydroxylating activity of each obtained bacterial cell lysate supernatant was evaluated by measuring the decrease in absorbance at a wavelength of 250 nm in the same manner as in Example 5. Furthermore, the same E. coli as in the well that showed the largest decrease in absorbance at a wavelength of 250 nm was cultured again in the same manner as above, and a plasmid was prepared in the same manner as in Example 1. The introduced mutation was identified by DNA sequencing of the plasmid according to a standard method.

[0187] Table 11 shows the mutated residues of the obtained mutants and the degree of decrease in absorbance at a wavelength of 250 nm.

[0188] As shown in Example 3, McPH-m3a-E124A, in which the E124A mutation has been introduced into McPH-m3a, has a higher L-pipecolic acid hydroxylation activity than McPH-m3a.

[0189] McPH-m7a to McPH-m7d showed a greater decrease in absorbance than the control McPH-m3a-E124A, indicating that they have greater L-pipecolic acid hydroxylation activity than McPH-m3a-E124A.

[0190] McPH-m7a to McPH-m7d are derived from McPH-m3a-E124A by further introducing the following mutations: D90S, I105L, L111F, D249D, and / or H278Y.

[0191] From this, it is considered that the D90S, I105L, L111F, D249D, and / or H278Y mutations contribute to the improvement of L-pipecolic acid hydroxylation activity. Furthermore, since all of McPH-m7a to McPH-m7d are obtained by further introducing the I105L, L111F, and H278Y mutations into McPH-m3a-E124A, it is considered that these mutations in particular greatly contribute to the improvement of L-pipecolic acid hydroxylation activity.

[0192]

[0193] (2) Preparation of transformants expressing the pKW plasmid-type mutant library and evaluation of L-pipecolic acid hydroxylation activity Using the pKW plasmid-type mutant library (pKW-McPH-cmb-lib) prepared in Example 4 and pKW32-McPH-m6a prepared in Example 1, Escherichia coli JM109 (Takara Bio Inc.) was transformed in the same manner as in Example 6(1), and the transformed Escherichia coli was inoculated onto an LB agar medium containing kanamycin as an antibiotic and cultured overnight at 30°C to obtain colonies of transformed Escherichia coli on the LB agar medium.

[0194] 200 μL of LB liquid medium containing 25 mg / L kanamycin as an antibiotic was dispensed into a 96-well deep well plate (manufactured by Whatman), and the colonies obtained by the above culture were inoculated one by one and cultured with shaking at 30°C as a preculture.

[0195] Next, 200 μL of LB medium containing 25 mg / L kanamycin, 0.4% glycerol, and 0.2 mmol / L IPTG was dispensed into another 96-well deep well plate (Whatman), and 5 μL of each culture solution obtained in the preculture was inoculated and cultured overnight with shaking at 32° C. to induce protein expression. A bacterial cell lysate supernatant was obtained from the resulting culture solution in the same manner as in Example 6(1).

[0196] The L-pipecolic acid hydroxylating activity of each obtained bacterial cell lysate supernatant was evaluated by measuring the decrease in absorbance at a wavelength of 250 nm in the same manner as in Example 5. Furthermore, the same E. coli as in the well that showed the largest decrease in absorbance at a wavelength of 250 nm was cultured again in the same manner as above, and a plasmid was prepared in the same manner as in Example 6(1) to identify the introduced mutation.

[0197] Table 12 shows the mutated residues of the obtained mutants and the degree of decrease in absorbance at a wavelength of 250 nm.

[0198] McPH-m7e, into which the mutations D90S, L105L, L111F, and E124A were introduced, showed a decrease in absorbance comparable to that of McPH-m6a, indicating that it exhibits L-pipecolic acid hydroxylation activity comparable to that of McPH-m6a.

[0199]

[0200] Example 7: Evaluation of stability of McPH wild-type enzyme and McPH mutants m3a, m6a, m7a, and m7b (1) Preparation of bacterial cell lysate supernatant for stability evaluation and heat treatment Using pET-McPH (wild-type), pET-McPH-m3a, and pET-McPH-m6a prepared in Example 1, and pET-McPH-m7a and pET-McPH-m7b obtained in Example 6, each enzyme solution was obtained in the same manner as in Example 2.

[0201] The protein concentration of each of the obtained enzyme solutions was measured using Bio-Rad's Quick Start (registered trademark) Bradford 1x Dye Reagent according to the attached protocol, and each solution was diluted with 0.1 mol / L MES (pH 7.0) to a protein content of 2.6 g / L. 0.12 mL of each solution was dispensed into 2.0 mL plastic tubes and heat-treated by holding each solution at 4°C, 15°C, 25°C, 35°C, 45°C, 55°C, and 60°C for 1 hour. Then, the solution was left to stand on ice to prepare enzyme solutions for stability evaluation.

[0202] (2) Evaluation of Stability of McPH Mutants In a 2.0 mL plastic tube, 20 mmol / L of L-pipecolic acid, 40 mmol / L of αKG, 25 mmol / L of L-ascorbic acid, and 0.5 mmol / L of FeSO were added. ４ , 2 mmol / L sodium citrate, 100 mmol / L MES buffer (pH 7.0), and each enzyme solution for stability evaluation obtained in (1) above were added to prepare reaction solutions. For each enzyme solution for stability evaluation, 60 μL was added for McPH (wild type), 30 μL for McPH mutant m3a, and 15 μL for other McPH mutants. 0.1 mL of each resulting reaction solution was shaken at 15°C for 30 minutes. Then, 0.05 mL of 1 mol / L hydrochloric acid was added to each reaction solution to stop the reaction, followed by 0.05 mL of 1 mol / L sodium hydroxide (NaOH) and 2 mmol / L copper sulfate (CuSO ４) was added, and the mixture was centrifuged to obtain a supernatant. The concentration of hydroxy-L-pipecolic acid contained in the obtained supernatant was measured under the HPLC analysis conditions described in Example 3, and the L-pipecolic acid hydroxylating activity was evaluated in the same manner as in Example 3. The L-pipecolic acid hydroxylating activity calculated here was evaluated as the relative activity (%) before and after heat treatment, relative to the activity when each enzyme solution for stability evaluation was kept at 4°C for 1 hour. The amino acid mutation sites of the evaluated McPH mutants are shown in Table 13, and the evaluation results of the relative activity are shown in Figure 3.

[0203]

[0204] As is clear from Figure 3, the relative activity at a heat treatment temperature of 35°C was such that McPH (wild-type) showed almost no activity and McPH-m3a was reduced to approximately 20%, whereas McPH-m6a and McPH-m7b had a relative activity of approximately 60%, and the McPH mutant m7a had a relative activity of approximately 140%.

[0205] As described above, McPH-m3a exhibits higher heat resistance compared to McPH (wild type), and it is thought that the introduced mutations F5Y, C23A, and D282E contribute to improved stability.

[0206] Furthermore, since McPH-m6a exhibits higher thermostability than McPH-m3a, it is believed that the introduced mutations L111F and H278Y contribute to improved stability.Similarly, since McPH-m7a exhibits higher thermostability than McPH-m6a, it is believed that the introduced mutations I105L and E124A also contribute to improved stability.

[0207] On the other hand, when comparing McPH-m7a and McPH-m7b, McPH-m7a shows higher heat resistance, so it is thought that D90S introduced in McPH-m7b does not contribute to improved stability.

[0208] Example 8: Preparation of saturation mutant library using pKW32-McPH-m7a or pKW32-McPH-m7e as template and evaluation of L-pipecolic acid hydroxylation activity (1) Preparation of plasmids pKW32-McPH-m7a and pKW32-McPH-m7c Using the plasmid pET-McPH-m7a obtained in Example 6 as a template, a portion of the McPH gene was amplified by PCR using primers of SEQ ID NO: 104 and SEQ ID NO: 105.

[0209] Furthermore, a DNA fragment was obtained by amplifying the vector portion containing the N-terminus and C-terminus of the McPH gene of the plasmid pKW32-McPH-m6a by PCR using the plasmid pKW32-McPH-m6a obtained in Example 1 as a template and the primers of SEQ ID NO: 106 and 107.

[0210] The two resulting DNA fragments were ligated using Gibson Assembly Master Mix (New England BioLabs) according to the attached protocol, and a plasmid named pKW32-McPH-m7a was prepared in the same manner as in Example 4(1). Further, pKW32-McPH-m7c was obtained in the same manner as above using the plasmid pET-McPH-m7c obtained in Example 6 as a template (Table 14).

[0211]

[0212] (2) Preparation of a Saturation Mutant Library Using pKW32-McPH-m7a as a Template Using the plasmid pKW32-McPH-m7a obtained in Example 8(1) as a template, site-specific saturation mutagenesis was performed on the amino acid at SEQ ID NO: 132 as follows to prepare a saturation mutant library.

[0213] Specifically, a DNA fragment in which a mutation was introduced into the 132nd amino acid of SEQ ID NO: 2 was amplified by PCR using the plasmid pKW32-McPH-m7a prepared in Example 8(1) as a template and the primers of SEQ ID NO: 112 and SEQ ID NO: 88.

[0214] Furthermore, other portions of the plasmid were amplified by PCR using the primers of SEQ ID NO: 113 and SEQ ID NO: 75 to obtain DNA fragments.

[0215] The two resulting DNA fragments were ligated using Gibson Assembly Master Mix (New England Biolabs) according to the attached protocol, and the resulting DNA solution was used to transform Escherichia coli DH5α (Takara Bio) according to the attached protocol, and the transformed strain was inoculated into LB liquid medium containing kanamycin as an antibiotic. Plasmids were prepared from the resulting strains using the same miniprep method as above, and a mutant library (pKW32-McPH-strn132) in which a saturation mutation was introduced at amino acid 132 of SEQ ID NO:2 was obtained (Table 15).

[0216]

[0217] (3) Evaluation of L-pipecolic acid hydroxylation activity of saturated mutants. Cell lysate supernatants were obtained from the mutant library prepared in Example 8(2) and pKW32-McPH-m7a prepared in Example 8(1) in the same manner as in Example 6(1).

[0218] For the McPH mutant library McPH-strn132, the absorbance at a wavelength of 250 nm was measured in the same manner as in Example 5(3), and the L-pipecolic acid hydroxylation activity was evaluated using McPH-m7a as a control.

[0219] The sequence of each mutant obtained and the degree of decrease in absorbance at a wavelength of 250 nm are shown in Table 16. In Table 16, the values ​​in parentheses represent standard deviations.

[0220]

[0221] As is clear from Table 16, the McPH mutants (CG3, CF10) in which the A132Q or A132K mutation was introduced into McPH-m7a exhibited higher L-pipecolic acid hydroxylation activity than the control McPH-m7a. This suggests that the A132Q or A132K mutation contributes to improved L-pipecolic acid hydroxylation activity.

[0222] Example 9: Effect of mutations at positions 132 and 249 of McPH on stability To verify the effect of mutations at positions 132 and 249 of McPH on stability, the stabilities of the mutants obtained in Example 8 were compared.

[0223] Using pKW32-McPH-m7a and pKW32-McPH-m7c obtained in Example 8(1), and pKW32-McPH-m7a-A132Q(CG3) and pKW32-McPH-m7a-A132K(CF10) obtained in Example 8(3), bacterial cell lysate supernatants were obtained in the same manner as in Example 5. 0.04 mL of each of the obtained bacterial cell lysate supernatants was dispensed into 2.0 mL plastic tubes, and each bacterial cell lysate supernatant was kept for 1 hour at each temperature of 20°C, 25°C, 30°C, 34°C, 38°C, 42°C, 46°C, and 50°C, and used as bacterial cell lysate supernatants for stability evaluation.

[0224] For each of the bacterial cell lysate supernatants for stability evaluation, the L-pipecolic acid hydroxylating activity of each bacterial cell lysate supernatant for stability evaluation was measured in the same manner as in Example 5 (3), and the stability was evaluated by determining the relative value of the L-pipecolic acid hydroxylating activity at each holding temperature, with the L-pipecolic acid hydroxylating activity when held at 20°C being defined as 100%. For each McPH mutant, the amino acid mutation sites are shown in Table 17, and the relative value of the L-pipecolic acid hydroxylating activity at each holding temperature is shown in Table 18.

[0225]

[0226]

[0227] As is clear from Table 18, the mutants into which the A132Q or S249D mutation was introduced maintained more activity than McPH-m7a at holding temperatures of 25°C to 42°C, and in particular, maintained more activity than McPH-m7a when held at relatively high temperatures such as 34°C, 38°C, and 42°C. Furthermore, the mutants into which the A132Q or S249D mutation was introduced showed higher relative values ​​when held at temperatures of 25°C to 38°C than when held at a temperature of 20°C. Furthermore, the mutants into which the A132K mutation was introduced maintained more activity than McPH-m7a at holding temperatures of 46°C to 50°C.

[0228] Example 10: Improvement of stability by N140S and N140M mutations (1) Preparation of plasmid pKW32-McPH-m8e Using pKW32-McPH-m7e obtained in Example 6(2) as a template, a DNA fragment in which serine at position 249 of SEQ ID NO: 2 was substituted with aspartic acid was amplified by PCR using the primers of SEQ ID NO: 123 and SEQ ID NO: 88 in the same manner as in Example 4, and a DNA fragment in which the other portion of the plasmid was amplified was obtained by PCR using the primers of SEQ ID NO: 124 and SEQ ID NO: 75. From the two obtained DNA fragments, a plasmid in which serine at position 249 of SEQ ID NO: 2 in pKW-McPH-m7e was substituted with aspartic acid was prepared in the same manner as in Example 5. Furthermore, using the obtained plasmid as a template, a DNA fragment in which alanine at position 132 of SEQ ID NO:2 was substituted with glutamine was amplified by PCR using the primers of SEQ ID NO:125 and SEQ ID NO:75, and a DNA fragment in which the other portion of the plasmid was amplified was obtained by PCR using the primers of SEQ ID NO:126 and SEQ ID NO:88. From the two obtained DNA fragments, a plasmid in which alanine at position 132 of SEQ ID NO:2 was substituted with glutamic acid and serine at position 249 of SEQ ID NO:2 was substituted with aspartic acid in pKW-McPH-m7e was prepared in the same manner as in Example 5, and this plasmid was designated pKW32-McPH-m8e.

[0229] (2) Preparation of a saturation mutant library at amino acid 140 using pKW32-McPH-m8e as a template Using pKW32-McPH-m8e prepared in Example 10(1) as a template, a DNA fragment in which a mutation was introduced at amino acid 140 of SEQ ID NO:2 was amplified by PCR using primers of SEQ ID NO:127 and SEQ ID NO:75, in the same manner as in Example 5, and a DNA fragment in which other portions of the plasmid were amplified by PCR using primers of SEQ ID NO:128 and SEQ ID NO:88. From the two resulting DNA fragments, a mutant library (pKW32-McPH-strn140) in which a saturation mutation was introduced at amino acid 140 of SEQ ID NO:2 was prepared in the same manner as in Example 5.

[0230] (3) Evaluation of L-pipecolic acid hydroxylation activity of the saturation mutant library For the mutant library prepared in Example 10(2), cell lysate supernatants were obtained from 168 different McPH mutant-expressing strains in the same manner as in Example 9, and each was kept at 20°C and 45°C for 1 hour to prepare cell lysate supernatants for stability evaluation.

[0231] The L-pipecolic acid hydroxylating activity of each bacterial cell lysis supernatant for stability evaluation was measured in the same manner as in Example 5 (3), and the stability was evaluated by determining the relative value of the L-pipecolic acid hydroxylating activity at each temperature, with the L-pipecolic acid hydroxylating activity when maintained at 20°C being defined as 100%. For McPH mutants with high relative values ​​of L-pipecolic acid hydroxylating activity, the 140th amino acid in SEQ ID NO: 2, the decrease in absorbance at 250 nm, and the relative value of the L-pipecolic acid hydroxylating activity when maintained at 45°C to that when maintained at 20°C are shown in Table 19.

[0232]

[0233] As is clear from Table 19, the relative values ​​of L-pipecolic acid hydroxylation activity when maintained at 45°C relative to when maintained at 20°C were significantly higher in the McPH mutants CB7 and BA2 in which the 140th amino acid in SEQ ID NO: 2 was substituted with serine, and BE7 in which the 140th amino acid in SEQ ID NO: 2 was substituted with methionine.

[0234] (4) Confirmation of the stability effect of McPH-m8e-N140S and McPH-m8e-N140M The McPH mutants CB7 and BA2 obtained in Example 10(3) were designated McPH-m8e-N140S, and BE7 was designated McPH-m8e-140M.

[0235] For McPH-m8e-N140S and McPH-m8e-N140M, the same procedure as in Example 10 (3) was used to measure the L-pipecolic acid hydroxylation activity of each bacterial cell lysate supernatant for stability evaluation, except that the incubation temperature was changed to 20 ° C., 25 ° C., 30 ° C., 34.75 ° C., 41 ° C., 44.25 ° C., 50.75 ° C., and 55 ° C. The L-pipecolic acid hydroxylation activity of each bacterial cell lysate supernatant for stability evaluation was measured by the same method. The L-pipecolic acid hydroxylation activity of McPH-m8e-N140S and McPH-m8e-N140M when kept at 20 ° C. was set to 100%, and the relative value of the L-pipecolic acid hydroxylation activity at each incubation temperature was calculated to evaluate stability. The results are shown in Table 20.

[0236]

[0237] As is clear from Table 20, McPH-m8e-N140S, into which the N140S mutation was introduced, maintained more activity than McPH-m8e when the holding temperature was 41°C to 55°C. Furthermore, McPH-m8e-N140M, into which the N140M mutation was introduced, maintained more activity than McPH-m8e when the holding temperature was 25°C to 55°C. Furthermore, the McPH mutants into which the N140S or N140M mutation was introduced maintained significantly more activity than m8e, even when held at relatively high temperatures such as 41°C or 44.25°C.

[0238] From this, it is considered that the N140S and N140M mutations contribute to improved stability.

Claims

1. An L-pipecolic acid hydroxylase comprising a polypeptide having any of the following amino acid sequences (1) to (3): (1) an amino acid sequence represented by SEQ ID NO: 2 in which one or more of the 111th, 105th, 124th, 132nd, 140th, 249th, and 278th amino acids are substituted with other amino acids; (2) an amino acid sequence in which the amino acid sequence of (1) has been modified with amino acids other than the substituted amino acids, and which has a sequence identity of 90% or more with the amino acid sequence of (1); (3) an amino acid sequence in which the amino acid sequence of (1) has one to several amino acid substitutions, deletions, and / or additions with respect to the amino acids other than the substituted amino acids.

2. The L-pipecolic acid hydroxylase according to claim 1, wherein the amino acid sequence (1) is an amino acid sequence having one or more amino acid substitutions selected from the following amino acid substitutions (a) to (g) in the amino acid sequence shown in SEQ ID NO: 2: (a) substitution of isoleucine at position 105 with leucine; (b) substitution of leucine at position 111 with phenylalanine; (c) substitution of glutamic acid at position 124 with alanine; (d) substitution of alanine at position 132 with glutamine or lysine; (e) substitution of asparagine at position 140 with serine or methionine; (f) substitution of serine at position 249 with aspartic acid; and (g) substitution of histidine at position 278 with tyrosine.

3. The L-pipecolic acid hydroxylase according to claim 1 or 2, wherein the amino acid sequence (1) is an amino acid sequence in which one or more of the amino acids at positions 5, 23, and 282 in the amino acid sequence shown in SEQ ID NO: 2 are further substituted with other amino acids.

4. The L-pipecolic acid hydroxylase according to claim 3, wherein the amino acid sequence (1) is the amino acid sequence shown in SEQ ID NO: 2, and further comprises one or more amino acid substitutions selected from the following amino acid substitutions (p) to (r): (p) substitution of phenylalanine at position 5 with tyrosine; (q) substitution of cysteine ​​at position 23 with alanine; (r) substitution of aspartic acid at position 282 with glutamic acid.

5. The L-pipecolic acid hydroxylase according to any one of claims 1 to 4, wherein the amino acid sequence of (1) is an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 52, 54, 118, 120 and 122.

6. A nucleic acid encoding the amino acid sequence of the L-pipecolic acid hydroxylase according to any one of claims 1 to 5.

7. The nucleic acid according to claim 6, wherein the nucleic acid comprises the nucleotide sequence shown in (4) or (5) below: (4) a nucleotide sequence having one to several nucleotide substitutions, deletions, and / or additions in the nucleotide sequence shown in SEQ ID NO: 1; (5) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO:

1.

8. A method for producing hydroxy-L-pipecolic acid, comprising contacting L-pipecolic acid with the L-pipecolic acid hydroxylase according to any one of claims 1 to 5, a microorganism or cell capable of producing said enzyme, a processed product of said microorganism or cell, and / or a culture solution containing said enzyme obtained by culturing said microorganism or cell, to produce hydroxy-L-pipecolic acid.

9. The method for producing hydroxy-L-pipecolic acid according to claim 8, wherein the microorganism or cell is a microorganism or cell transformed with the nucleic acid according to claim 6 or 7.

10. A recombinant vector comprising the nucleic acid of claim 6 or 7.

11. A transformant comprising the recombinant vector according to claim 10.

Images