L-lysine position-5 hydroxylase, and method for producing 5-hydroxy-l-lysine using same

Abstract

The present invention addresses the problem of providing a novel method for inexpensively producing 5-hydroxy-L-lysine with high yield and at low cost. As a solution for the problem, the present invention provides: a novel L-lysine position-5 hydroxylase; an enzyme agent composition for producing 5-hydroxy-L-lysine, the composition containing the hydroxylase; and a method for producing 5-hydroxy-L-lysine using the enzyme agent composition.

Description

L-lysine 5-hydroxylase and method for producing 5-hydroxy-L-lysine using the same

[0001] The present invention relates to an L-lysine 5-hydroxylase, a method for producing 5-hydroxy-L-lysine, and a catalyst for producing 5-hydroxy-L-lysine.

[0002] 5-Hydroxy-L-lysine is useful as an intermediate for pharmaceuticals. For example, it is known that 5-hydroxy-L-lysine can be used as an intermediate for Bengamide B, which has antitumor activity. It has also been reported that 5-hydroxy-L-lysine can be used as a raw material for 5-hydroxy-L-pipecolic acid (Non-Patent Documents 1 and 2). 5-Hydroxy-L-pipecolic acid is known to be useful as a precursor for antibacterial agents (Patent Document 1).

[0003] Methods for synthesizing 5-hydroxy-L-lysine have been reported, for example, in Non-Patent Documents 3 to 6. However, all of these synthetic methods involve long or complicated production steps or require expensive raw materials or catalysts, and therefore, there has been a demand for a method for inexpensively producing 5-hydroxy-L-lysine in high yield at low cost.

[0004] Patent Document 2 reports that 2-ketoglutarate-dependent dioxygenase has an activity of hydroxylating lysine at the 3- or 4-position, and also reports an efficient method for producing 3-hydroxy-L-lysine and / or 4-hydroxy-L-lysine using the enzyme.

[0005] From Patent Document 2, it can be predicted that if an L-lysine hydroxylase having hydroxylation activity at the 5-position of L-lysine can be found, a method for producing 5-hydroxy-L-lysine from L-lysine can be established.

[0006] Here, Non-Patent Document 7 suggests that by knocking out a part of the arazopeptin biosynthetic gene cluster (hereinafter referred to as the "azp gene cluster") in the Streptacidiphilus griseoplanus JCM4300 strain, the AzpK protein of SEQ ID NO: 136 or SEQ ID NO: 138 (the base sequences of DNA encoding the amino acid sequences of these proteins are SEQ ID NOs: 135 and 137, respectively) encoded by the azpK gene in the azp gene cluster may have the activity of hydroxylating L-lysine at the 5-position.

[0007] Therefore, by combining the findings of Non-Patent Document 2 and Non-Patent Document 7, it is possible that a polypeptide having an activity of hydroxylating the 5-position of L-lysine may exist among polypeptides encoded by the azpK gene or an azpK gene homologue having high sequence identity thereto, and it can be easily imagined by those skilled in the art that by using such a polypeptide, a method for producing 5-hydroxy-L-lysine from L-lysine can be established.

[0008] However, Non-Patent Document 7 reports that the AzpK protein of SEQ ID NO: 136 or 138 has no sequence similarity in amino acid sequence with known hydroxylases, and the reaction mechanism is unknown, and that although the AzpK protein was obtained from the azpK gene and its L-lysine 5-hydroxylation activity was evaluated, the activity could not be detected.

[0009] Furthermore, Non-Patent Document 8 reports an L-lysine halogenating (chlorinating) enzyme called BesD, which has sequence similarity to the amino acid sequence of the polypeptide of the present invention described below, but does not describe the function of the polypeptide of the present invention.

[0010] Patent No. 4590981 Patent No. 6476110

[0011] Yasuda et al., Tetrahedron Asymmetry, 2006, 17, 1775; Tsotsou et al., Biochemie, 2007, 89, 591; Allevi et al., Tetrahedron Asymmetry, Vol. 11, Issue 15, 2000, 3151-3160; Guo et al., Org. Biomol. Chem., 2014, 12, 7310-7317; Nieuwendijk et al., Eur. J. Org. Chem. 2000, 3683-3691; Johannes et al., J. Org. Chem. 2013, 78, 12809-12813 Kawai et al., Angew. Chem. Int. Ed. 2021, 60, 10319-10325 Neugebauer et al., Nat. Chem. Bio., Vol. 15, Oct. 2019, 1009-1016

[0012] As described above, although methods for producing 5-hydroxy-L-lysine have been known, there has been a demand for a method for producing 5-hydroxy-L-lysine inexpensively with higher yield and at lower cost. Furthermore, a method for producing 5-hydroxy-L-lysine from L-lysine using an L-lysine 5-hydroxylase, which could be considered an efficient production method, has not been technically realized because the L-lysine 5-hydroxylase has not yet been discovered.

[0013] That is, an object of the present invention is to provide a method for inexpensively producing 5-hydroxy-L-lysine at high yield and at low cost, particularly to discover an L-lysine 5-hydroxylase capable of producing 5-hydroxy-L-lysine from L-lysine, and to provide a novel method for inexpensively producing 5-hydroxy-L-lysine at high yield and at low cost using the same.

[0014] In order to solve the above-mentioned problems, the present inventors conducted extensive research on genes having L-lysine 5-hydroxylase activity based on the information in Non-Patent Document 7. As a result, they discovered that 2-oxoglutarate-dependent hydroxylases, the function of which had not previously been reported as isolated proteins, possess L-lysine 5-hydroxylase activity, and further identified amino acid sequence motifs important for the expression of L-lysine 5-hydroxylase. They then prepared transformants using DNA encoding these polypeptides, and found that 5-hydroxy-L-lysine can be produced at high optical purity and high concentration by allowing the transformant cells, preparations thereof, and / or culture medium to act on L-lysine. The present invention was achieved based on these findings. The gist of the present invention is as follows:

[0015] [1] An L-lysine 5-hydroxylase comprising a polypeptide shown in the following (A), (B), (C), (D), (E), or (F): (A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134; (B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having L-lysine 5-hydroxylation activity; (C) a polypeptide having an amino acid sequence which has 55% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and which has L-lysine 5-hydroxylase activity; (D) a polypeptide having an amino acid sequence which has 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and which has L-lysine 5-hydroxylation activity; (E) a polypeptide having an amino acid sequence having 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, and having L-lysine 5-hydroxylating activity; (F) a consecutive amino acid sequence motif "HGWHWDD";and any motif of a consecutive amino acid sequence selected from the group consisting of "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," and which retains L-lysine 5-hydroxylating activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). [2] DNA encoding the L-lysine 5-hydroxylating enzyme. [3] The DNA, which is the following (G), (H), (I), (J), (K), or (L): (G) a DNA containing the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133; (H) DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, in which one or more nucleotides have been substituted, deleted, and / or added; (I) DNA encoding a polypeptide having 55% or more sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having L-lysine 5-hydroxylating activity;(J) a DNA encoding a polypeptide having 50% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and having L-lysine 5-hydroxylating activity; (K) a DNA having 58% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and having L-lysine 5-hydroxylating activity; (L) DNA encoding a polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," and having activity of hydroxylating the 5-position of L-lysine (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). [4] A method for producing 5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide represented by the following (A), (B), (C), (D), (E), or (F), or a cell containing the polypeptide, a preparation of the cell, or a culture medium obtained by culturing the cell, to produce 5-hydroxy-L-lysine: (A) a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having L-lysine 5-hydroxylation activity; (C) a polypeptide having an amino acid sequence which has 55% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and which has L-lysine 5-hydroxylase activity; (D) a polypeptide having an amino acid sequence which has 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and which has L-lysine 5-hydroxylation activity; (E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 and having L-lysine 5-hydroxylating activity; (F) a consecutive amino acid sequence motif "HGWHWDD"; and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF";and having an activity of hydroxylating L-lysine at the 5-position (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). [5] The method for producing 5-hydroxy-L-lysine as described above, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase. [6] The method for producing 5-hydroxy-L-lysine, wherein the DNA is the following (G), (H), (I), (J), (K), or (L): (G) a DNA containing the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133; (H) DNA encoding a polypeptide having an L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, in which one or more nucleotides have been substituted, deleted, and / or added; (I) DNA encoding a polypeptide having 55% or more sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and having L-lysine 5-hydroxylating activity;(J) a DNA encoding a polypeptide having 50% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and having L-lysine 5-hydroxylating activity; (K) a DNA having 58% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and having L-lysine 5-hydroxylating activity; (L) DNA encoding a polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having L-lysine 5-hydroxylating activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V). [7] An enzyme composition having L-lysine 5-hydroxylating activity, comprising a polypeptide shown in (A), (B), (C), (D), (E) or (F) below, or a cell containing the polypeptide, a preparation of the cell or a culture medium obtained by culturing the cell: (A) a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134;(B) a polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having L-lysine 5-hydroxylation activity; (C) a polypeptide having an amino acid sequence which has 55% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and which has L-lysine 5-hydroxylase activity; (D) a polypeptide having an amino acid sequence which has 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and which has L-lysine 5-hydroxylation activity; (E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 and having L-lysine 5-hydroxylating activity; (F) a consecutive amino acid sequence motif "HGWHWDD"; and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF";and having an activity of hydroxylating the 5-position of L-lysine (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). [8] An S-selective L-lysine 5-hydroxylase comprising a polypeptide shown in (A-1), (B-1), (C-1), or (F-1) below: (A-1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22; (B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylation activity; (C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having S-selective L-lysine 5-hydroxylase activity; (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). [9] A DNA encoding the S-selective L-lysine 5-hydroxylase.

[10] The DNA, which is the following (G-1), (H-1), (I-1), or (L-1): (G-1) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21; (H-1) a DNA having a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having L-lysine 5-hydroxylating activity; (I-1) a DNA having 55% or more sequence identity to the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having S-selective L-lysine 5-hydroxylating activity;(L-1) DNA encoding a polypeptide having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

[11] A method for producing (2S,5S)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-1), (B-1), (C-1), or (F-1), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells, to produce (2S,5S)-5-hydroxy-L-lysine: (A-1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22; (B-1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 in which one or more amino acids have been deleted, substituted, and / or added, and having S-selective L-lysine 5-hydroxylation activity; (C-1) A polypeptide having an amino acid sequence that has 55% or more sequence identity to the full-length amino acid sequence of SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 and having S-selective L-lysine 5-hydroxylase activity; (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and retaining S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

[12] The method for producing (2S,5S)-5-hydroxy-L-lysine, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase.

[13] The method for producing (2S,5S)-5-hydroxy-L-lysine, wherein the DNA is the following (G-1), (H-1), (I-1), or (L-1): (G-1) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21;(H-1) DNA encoding a polypeptide having the activity of hydroxylating L-lysine at the 5th position, which has a base sequence in which one or more bases have been substituted, deleted, and / or added in the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21; (I-1) DNA having a sequence identity of 55% or more to the polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, which encodes a polypeptide having S-selective L-lysine 5-hydroxylating activity; (L-1) DNA encoding a polypeptide having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylating activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

[14] An enzyme composition comprising a polypeptide shown in (A-1), (B-1), (C-1) or (F-1) below, or a cell containing the polypeptide, a preparation of the cells or a culture medium obtained by culturing the cells, and having S-selective L-lysine 5-hydroxylation activity: (A-1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22; (B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity; (C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity;(F-1) A polypeptide having the consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF" and having S-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

[15] An R-selective L-lysine 5-hydroxylase comprising a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2), or (F-2): (A-2) a polypeptide having the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134; (B-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having R-selective L-lysine 5-hydroxylation activity; (D-2) a polypeptide having an amino acid sequence that has 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity; (E-2) a polypeptide having an amino acid sequence that has 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, and having R-selective L-lysine 5-hydroxylation activity;(F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

[16] DNA encoding the R-selective L-lysine 5-hydroxylase.

[17] The DNA, which is the following (G-2), (H-2), (J-2), (K-2), or (L-2): (G-2) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133; (H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, in which one or more nucleotides have been substituted, deleted, and / or added; (J-2) DNA encoding a polypeptide having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and having R-selective L-lysine 5-hydroxylation activity;(K-2) DNA encoding a polypeptide having 58% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and having R-selective L-lysine 5-hydroxylating activity; (L-2) DNA encoding a polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF" (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

[18] A method for producing (2S,5R)-5-hydroxy-L-lysine, comprising contacting L-lysine with a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2), or (F-2), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells, to produce (2S,5R)-5-hydroxy-L-lysine: (A-2) a polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134; (B-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having R-selective L-lysine 5-hydroxylation activity;(D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity to the full-length amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity; (E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity; (F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD"; and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

[19] The method for producing (2S,5R)-5-hydroxy-L-lysine as described above, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-hydroxylase.

[20] The method for producing (2S,5R)-5-hydroxy-L-lysine, wherein the DNA is the following (G-2), (H-2), (J-2), (K-2), or (L-2): (G-2) a DNA containing the nucleotide sequence set forth in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133;(H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, in which one or more nucleotides have been substituted, deleted, and / or added; (J-2) DNA having 50% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (K-2) DNA having 58% or more sequence identity to a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (L-2) a consecutive amino acid sequence motif "HGWHWDD"; and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF", and "HETNNXLF";and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

[21] An enzyme composition having R-selective L-lysine 5-hydroxylation activity, comprising a polypeptide shown in the following (A-2), (B-2), (D-2), (E-2), or (F-2), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells: (A-2) a polypeptide having the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134; (B-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having R-selective L-lysine 5-hydroxylation activity; (D-2) a polypeptide having an amino acid sequence that has 50% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity; (E-2) a polypeptide having an amino acid sequence that has 58% or more sequence identity to the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, and having R-selective L-lysine 5-hydroxylation activity;(F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity (wherein X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V);

[0016] The present invention can also employ the following configurations.

[22] A method for hydroxylating L-lysine, comprising contacting L-lysine with the polypeptide shown in (A), (B), (C), (D), (E), or (F), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells, to hydroxylate the 5-position of L-lysine.

[23] Use of the polypeptide shown in (A), (B), (C), (D), (E), or (F), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells, as an L-lysine 5-hydroxylase composition.

[24] Use of the polypeptide shown in (A), (B), (C), (D), (E), or (F), or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells, in the production of an L-lysine 5-hydroxylase composition.

[0017] According to the present invention, it is possible to provide an L-lysine 5-hydroxylase and an enzyme composition for producing 5-hydroxy-L-lysine. Furthermore, it is possible to provide a method for inexpensively producing 5-hydroxy-L-lysine from L-lysine in high yield and at low cost. Furthermore, it is possible to provide a method for producing the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine by selectively using different L-lysine 5-hydroxylase enzymes.

[0018] 1 is a diagram comparing azp gene clusters. This diagram shows phylogenetic tree classification of homologs of the azpK2 gene and the Pp_azpK2 gene. This diagram shows the results of LC-MS detection of reaction products when AzpK2 is used in Example 5. This diagram shows the results of HPLC analysis of Fmoc-modified 5-hydroxylysine and 5-oxolidine in Example 7. This diagram shows the results of detection of 5-oxolidine in combination with AzpK2 Homolog 4-1 and an oxidase in Example 10. This diagram shows the results of activity evaluation of S-selective AzpK2 homologs using genetically modified Escherichia coli in Example 13. This diagram shows the results of examining cell disruption conditions in Example 15. This diagram shows the results of examining the pH of the reaction solution in Example 15. This diagram shows the results of examining the substrate concentration in Example 15. This diagram shows the results of examining the substrate concentration in Example 15. This diagram shows the results of identity comparison of the amino acid sequences of L-lysine 5-hydroxylase in Example 17. 1 shows the results of an identity comparison of the amino acid sequences of L-lysine 5-hydroxylase in Example 17. 2 shows the results of model construction by X-ray structural analysis of AzpK2 protein crystals in Example 18. 3 shows the results of an amino acid identity comparison between AzpK2 and BesD in Example 19. 4 shows the results of three-dimensional structure prediction of AzpK2 and BesD by AlphaFold2 in Example 20. 5 shows the results of activity evaluation of amino acid-substituted AzpK2 mutants in Example 21. 6 shows a logo plot corresponding to amino acids 131 to 145 of AzpK2 and an AzpK2 homolog in Example 22. 7 shows a logo plot corresponding to amino acids 231 to 245 of AzpK2 and an AzpK2 homolog in Example 22.

[0019] The terms used in this specification are explained below. As used herein, "L-lysine" refers to L-lysine with CAS Registry Number 56-87-1.

[0020] As used herein, the terms "3rd position," "4th position," and "5th position" refer to the positions of the carbon atoms at "3rd position," "4th position," and "5th position" of L-lysine, respectively, and refer to the positions of the carbon atoms numbered as shown in formula (IV) below.

[0021]

[0022] As used herein, "5-position of L-lysine" refers to the carbon atom located at number "5" in the formula (IV).

[0023] As used herein, "5-hydroxy-L-lysine" means either one or both of "(2S,5S)-5-hydroxy-L-lysine" or "(2S,5R)-5-hydroxy-L-lysine."

[0024] As used herein, "5-hydroxylysine" means any one, or two to four, selected from the group consisting of "(2S,5S)-5-hydroxy-L-lysine," "(2S,5R)-5-hydroxy-L-lysine," "(2R,5S)-5-hydroxy-D-lysine," and "(2R,5R)-5-hydroxy-D-lysine."

[0025] As used herein, "hydroxylation" refers to a reaction that converts a specific C--H bond in a compound into a C--OH bond (a reaction that adds a hydroxyl group to a specific carbon atom in a compound).

[0026] As used herein, "hydroxylation activity" refers to catalytic activity in hydroxylation per unit mass (e.g., milligram) of protein, dry cell mass, or immobilized catalyst mass.

[0027] As used herein, the term "L-lysine 5-hydroxylation activity" refers to the ability to add a hydroxyl group to the carbon atom at position 5 of L-lysine.

[0028] Such activity can be confirmed, for example, by directly or indirectly confirming the production of 5-hydroxy-L-lysine in a reaction system containing L-lysine as a substrate and further containing 2-oxoglutaric acid, using L-lysine 5-hydroxylase as a catalyst, or cells containing it, a preparation of the cells, or a culture medium obtained by culturing the cells, as described in the Examples below.

[0029] Methods for directly detecting 5-hydroxy-L-lysine include subjecting 5-hydroxy-L-lysine to HPLC for separation and detection by UV absorption, but 5-hydroxy-L-lysine has low UV absorbance and is difficult to detect. Methods for indirectly detecting 5-hydroxy-L-lysine include a method in which a copper complex of 5-hydroxy-L-lysine is formed using a mobile phase containing 5-hydroxy-L-lysine and copper ions, followed by HPLC to detect the UV absorption spectrum of the copper complex, or a method in which a UV-absorbing derivative such as a 9-fluorenylmethyloxycarbonyl (hereinafter sometimes referred to as "Fmoc") group is introduced into the amino group of 5-hydroxy-L-lysine, the derivative is subjected to HPLC for separation, and the separated derivative is detected and quantified using UV absorption or mass spectrometry (MS).

[0030] In addition, the stereochemistry of the hydroxyl group at the 5-position of 5-hydroxy-L-lysine can be determined by the following formula (V):

[0031]

[0032] One example of such a method is to use 5-hydroxy-L-lysine oxidase to stereoselectively oxidize the hydroxyl group of (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine to produce 5-oxo-L-lysine, and then detect NADH, which is produced in an equimolar amount to 5-oxo-L-lysine during the production, as shown in the reaction shown in Figure 1. This method makes it possible to detect 5-hydroxy-L-lysine and distinguish the stereochemistry of the hydroxyl group of 5-hydroxy-L-lysine.

[0033] Also, the compound represented by the following formula (VI):

[0034]

[0035] Another example is a method for determining the hydroxyl group of 5-hydroxy-L-lysine by producing 5-hydroxy-L-pipecolic acid from 5-hydroxy-L-lysine and identifying the hydroxyl group at the 5-position of 5-hydroxy-L-pipecolic acid, as shown in the reaction shown in Figure 1. Methods for producing 5-hydroxy-L-pipecolic acid from 5-hydroxy-L-lysine include a method using L-lysine / L-ornithine cyclodeaminase, as well as a method using a combination of multiple oxidoreductases such as amino acid dehydrogenase and imine reductase.

[0036] As used herein, "one unit of enzyme activity," "one unit of activity," or "U" refers to the hydroxylation activity required to produce 1 μmol of 5-hydroxylysine per minute at a particular temperature.

[0037] The one-letter and three-letter codes for amino acids used in this specification follow the "Guidelines for the preparation of specifications, etc. including nucleotide or amino acid sequences (corresponding to ST.26)" (Japan Patent Office). Specifically, they are as follows:

[0038]

[0039] In this specification, when representing an amino acid residue in a protein (polypeptide), a combination of the three-letter code of an amino acid and a number represents the amino acid residue and its residue number in the protein. For example, "His139" represents histidine, the 139th amino acid residue in the protein. Furthermore, when a single-letter code of an amino acid is written before and after a number, it represents a substitution of an amino acid residue in the protein, with the original amino acid residue written before the number (residue number) and the substituted amino acid residue written after the number (residue number). For example, "R76A" represents a substitution of alanine for arginine, the 76th amino acid residue in the protein.

[0040] As used herein, the terms "signature motif," "amino acid motif," and "motif" refer to a common conserved structure shared by a group of enzymes (enzyme families) with a particular activity. These signature motifs and the like can be used to characterize or identify structurally related enzyme families that have similar enzymatic activity toward a particular group of substrates. These signature motifs and the like typically exist as a single contiguous amino acid sequence or as a collection of non-adjacent conserved motifs. The conserved motifs can also be represented by amino acid sequences.

[0041] As used herein, the term "gene cluster" refers to a collection of genes in which multiple gene sequences are physically close together and typically function in a coordinated manner to contribute to a specific biological process or physiological function.

[0042] These clusters typically interact with each other in biological processes such as gene regulation, transcription, and translation, and can affect specific cellular functions and biological pathways. However, the number, types, and order of genes that make up a gene cluster can vary between organisms.

[0043] As used herein, "percent identity" is an index showing the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, and can be determined, for example, by comparing two or more polypeptide sequences. Sequence identity refers to the percentage of matching amino acids or nucleotides shared between two polypeptide or polynucleotide sequences when the two sequences are optimally aligned. That is, identity can be calculated as follows: identity = (number of matching positions / total number of positions) x 100. "Identity" can be easily calculated by known methods, such as, but not limited to, those listed below.

[0044] Computational Molecular Biology (Lesk, A.M., ed.), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.), Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), Stockton Press, NY (1991).

[0045] Furthermore, percent identity can be calculated using publicly available computer programs, such as, but not limited to, the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisconsin; or the EMBOSS Open software suite (EMBL-EBI; Rice et al., Trends in Genetics 16(6) pp. 276-277 (2000)).

[0046] In this case, multiple alignment of sequences can be performed using the Clustal method of alignment (i.e., CLUSTALW; e.g., version 2.4) (Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res. 22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res 31(13):3497-500 (2003), available from the European Molecular Biology Laboratory through the European Bioinformatics Institute) using default parameters.

[0047] Also, suitable parameters for CLUSTALW protein alignment include GAP existence penalty = 15, GAP extension = 0.2, matrix = Gonnet (e.g., Gonnet250), protein ENDGAP = -1, protein GAP DIST = 4, and KTUPLE = 1. In one embodiment, either fast or slow alignment is used with default settings, with slow alignment being preferred. Alternatively, parameters using the CLUSTALW method (version 1.83) may be modified to also use KTUPLE = 1, GAP penalty = 10, GAP extension = 1, matrix = BLOSUM (e.g., BLOSUM64), window = 5, and TOP DIAGONALS SAVED = 5.

[0048] As used herein, "sequence analysis software" refers to a computer algorithm or software program useful for analyzing amino acid sequences or nucleotides. Sequence analysis software may be commercially available or independently developed. Examples of such sequence analysis software include, but are not limited to, the following:

[0049] GCG programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc. 1228 S. Park St., Madison, WI 53715 USA); CLUSTALW (e.g., Version 2.4; Thompson et al., Nucleic Acids Research, 22(22):4673-4680 (1994); the FASTA program incorporating the Smith-Waterman algorithm (W.R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY); Sequencher v. 4.05

[0050] In this specification, when sequence analysis software is used to analyze amino acid sequences or nucleotides, the analysis results are, in principle, based on the "default values" of the program.

[0051] That is, as used herein, "default values" refers to a set of values ​​or parameters established by the software manufacturer and originally loaded with the software when it is first initialized.

[0052] As used herein, "X-ray structural analysis" encompasses techniques for elucidating the molecular structure of proteins or other compounds, including protein preparation, purification, optimization of crystallization conditions, irradiation of crystals with X-rays, and analysis of the resulting data. One technique is shown below, but it is not limited to this. Protein preparation is a process of protein synthesis using living cells or acellular systems, and various methods are applied depending on the properties of the protein. Protein purification is commonly performed using ion exchange chromatography, in which the protein is passed through an ion exchange resin packed in a column. The interaction between the ion exchange resin and the ions in the solution separates and purifies the protein. Optimizing crystallization conditions involves first identifying the crystallization conditions through initial screening, and then adjusting the pH of the crystallization buffer and the amount of precipitant to find the optimal crystallization conditions. pH adjustment is an important factor affecting the charge state and stability of the protein, while precipitants control crystal growth and promote the formation of ideal crystals. Obtaining crystals under optimal conditions improves the accuracy of X-ray structural analysis and enhances the quality of the analysis results. By irradiating the obtained crystals with X-rays while maintaining a constant environment, an X-ray diffraction pattern is obtained, and by processing the obtained diffraction pattern using XDS software, phase information can be extracted. Furthermore, by using structural modeling software such as AutoBuild to build a three-dimensional structural model of the protein using the extracted phase information, a three-dimensional structural model of the protein can be constructed. During crystallization, in addition to the protein to be analyzed, reaction substrates, substrate analogs, coenzymes, etc. can also be crystallized together with the protein, which may reveal information important to the function of the protein, such as interactions with the substrate and reaction mechanisms.

[0053] As used herein, "protein structure prediction software" refers to software for predicting the three-dimensional structure of a protein based on its sequence information. Protein structure is extremely important in understanding its function and properties, but protein structures are extremely complex, and various modeling techniques and supporting software have been developed. Modeling techniques include, for example, comparative modeling and de novo modeling. Comparative modeling predicts the target structure by comparing a protein (target) whose structure is to be predicted with a protein whose structure has already been determined as a template. When the template and target have similar structures, the target structure can be estimated based on the structure of the template. This technique is particularly useful when the target and template are similar proteins. De novo modeling predicts the structure based on the amino acid sequence information of the protein without requiring existing structural information. Protein structure is believed to be determined by the amino acid sequence pattern, and de novo modeling analyzes this pattern to predict the structure. The AlphaFold2 used in the present invention is a type of protein structure prediction software, a hybrid structure prediction software that combines comparative modeling and de novo modeling. This software first constructs an initial model using a protein with a known structure as a template, and then modifies the model using de novo modeling to predict the structure with higher accuracy. AlphaFold2 is provided as open source, and the source code is available on GitHub. AlphaFold2 can be run by downloading this source code and building it on an appropriate personal computer. When using AlphaFold2, protein sequence information can be input in a format such as a PDB file or FASTA file to obtain the desired structure prediction information.

[0054] As used herein, "mM" represents mmol / L, and "μM" represents μmol / L. As used herein, "W / V %" represents weight / volume %. As used herein, "HEPES" represents 4-2-hydroxyethyl-1-piperazineethanesulfonic acid. As used herein, "MES" represents 2-morpholinoethanesulfonic acid. As used herein, "VC" represents L-ascorbic acid. As used herein, "5HL" represents 5-hydroxy-L-lysine.

[0055] The SEQ ID NOs and sequence abbreviations of the amino acid sequences of the obtained L-lysine 5-hydroxylase enzymes are listed in Table 1 herein. The SEQ ID NOs of the base sequences of the DNAs encoding the amino acid sequences are listed in Table 2.

[0056]

[0057]

[0058] The present invention will be described in detail below. 1. Search for the L-lysine 5-hydroxylase of the present invention The L-lysine 5-hydroxylase of the present invention was discovered as follows. Based on the information in Non-Patent Document 7, a search was conducted for a gene having L-lysine 5-hydroxylase activity. Specifically, based on the genetic information of a Streptacidiphilus griseoplanus strain (hereinafter sometimes referred to as an "S. griseoplanus strain") and a Kitasatospora azatica strain (hereinafter sometimes referred to as a "K. azatica strain") having the arazopeptin biosynthetic gene cluster described in Non-Patent Document 7, a search was conducted for a microorganism having an azp gene cluster.

[0059] As a result, as shown in Figure 1, it was found that the homologous clusters of the azp gene cluster in the Actinosynnema pretiosum strain (hereinafter sometimes referred to as "A. pretiosum strain") and the Actinosynnema mirum DSM 43827 strain (hereinafter sometimes referred to as "A. mirum strain") do not contain the azpK gene, but instead contain a 2-oxoglutarate-dependent hydroxylase gene of unknown function (indicated by * in Figure 1). This 2-oxoglutarate-dependent hydroxylase was named AzpK2, and the DNA encoding the AzpK2 protein was named the azpK2 gene.

[0060] A transformant was prepared using the azpK2 gene, and the activity of the AzpK2 protein to hydroxylate the 5-position of L-lysine was measured. It was found that the AzpK2 protein had the activity to selectively hydroxylate the 5-position of L-lysine in an S-selective manner.

[0061] Furthermore, a similar search for the biosynthetic genes (azpJ, azpK, azpL) present in the azp gene cluster for 6-diazo-5-oxo-L-norleucine revealed that in Pseudomonas psychotolerans NS383 strain (hereinafter sometimes referred to as "P. psychotolerans strain"), there is an azpK2 gene homologue with unknown function between the azpJ gene and the azpL gene (indicated by a star in Figure 1). This gene was named Pp_azpK2, and the protein encoded by the Pp_azpK2 gene was named Pp_AzpK2.

[0062] FIG. 1 is a diagram of the azp gene cluster, and the meanings of the symbols in FIG. 1 are as follows:

[0063]

[0064] Furthermore, a transformant was prepared using the Pp_azpK2 gene, and the activity of the Pp_AzpK2 protein to hydroxylate the 5-position of L-lysine was measured. It was found that the transformant had the activity of R-selectively hydroxylating the 5-position of L-lysine.

[0065] Furthermore, when 5-hydroxy-L-lysine was synthesized from L-lysine using transformants of DNA encoding the AzpK2 protein (azpK2 gene) and DNA encoding the Pp_AzpK2 protein (Pp_azpK2 gene), it was found that 5-hydroxy-L-lysine was synthesized at a high accumulated concentration.

[0066] Furthermore, homology searches were performed for the azpK2 gene and the Pp_azpK2 gene using databases such as the DNA Databank of JAPAN (DDBJ), and gene sequence information for 1000 homologous genes of each of the azpK2 gene and the Pp_azpK2 gene was obtained.

[0067] Next, overlapping homologous genes of the azpK2 gene and homologous genes of the Pp_azpK2 gene were eliminated to obtain gene sequence information of 1,115 homologous genes including the azpK2 gene and the Pp_azpK2 gene.

[0068] The amino acid sequences encoded by these homologous genes were classified based on a molecular phylogenetic tree created using the Neighbor-joining method, and were classified into 10 Clades. Each classified Clade was named Clades 1 to 10. Representative proteins encoded by the 1,115 homologous genes (including the azpK2 and Pp_azpK2 genes) classified into the 10 Clades are shown in Figure 2.

[0069] The L-lysine 5-hydroxylase activity of proteins belonging to these 10 Clades was examined, and it was found that proteins belonging to Clades 1 to 4 have L-lysine 5-hydroxylase activity. Furthermore, it was found that proteins belonging to Clades 1 have the activity of S-selectively hydroxylating the 5-position of L-lysine, and that proteins belonging to Clades 2 to 4 have the activity of R-selectively hydroxylating the 5-position of L-lysine. Of these 10 Clades, Clade 1 included the AzpK2 protein, Clade 10 included the Pp_AzpK2 protein, and Clade 5 included the L-lysine halogenase reported in Non-Patent Document 8.

[0070] Furthermore, the AzpK2 protein was subjected to X-ray structural analysis, three-dimensional structural prediction, comparative analysis with L-lysine halogenating enzymes belonging to Clade 5, and identity analysis with the amino acid sequences of Clades 1 to 5. As a result, it was found that the amino acid motifs consisting of amino acid residues 136 to 142 and the amino acid motifs consisting of amino acid residues 235 to 242 of the AzpK2 protein are important for the expression of L-lysine 5-hydroxylation activity.

[0071] Among 2-oxoglutarate-dependent dioxygenases, the signature motif associated with enzymes that mainly catalyze hydroxylation reactions is known to include the following two conserved motifs. The positions of the amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2). 1) His139-X140-Asp141: "HXD" motif 2) Tyr197-Arg216: "Y-R" motif (Unless otherwise specified herein, X represents any of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.)

[0072] The L-lysine 5-hydroxylase of the present invention has the above-mentioned signature motif and is classified as a 2-oxoglutarate-dependent dioxygenase having EC number (Enzyme Commission number) 1.14.11.

[0073] Using a conventionally known global alignment algorithm (i.e., sequence analysis software), two or more amino acid sequences representing enzymes having L-lysine 5-hydroxylation activity can be aligned and compared to determine whether they share a common signature motif.

[0074] Therefore, the amino acid sequences of proteins that were found to have L-lysine 5-hydroxylation activity were analyzed using CLUSTAL alignment (CLUSTALW) with SEQ ID NO: 2 as a reference sequence, and it was found that the L-lysine 5-hydroxylase of the present invention contains, in addition to the signature motif, the following consecutive amino acid sequence motifs 3) and 4): 3) His136-Gly137-Trp138-His139-Trp140-Asp141-Asp142: "HGWHWDD" (SEQ ID NO: 144) motif (motif of amino acids 136 to 142; common to Clade 1 to 4); 4) His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Phe242: "HETMEXLF" (SEQ ID NO: 145) motif (motif 1 of amino acids 235 to 242; common to Clade 1); His235-Gln236-Thr237-Met238-Asp239-X240-Leu241-Trp242: "HETMDXLW" (SEQ ID NO: 146) motif (motif 2 of amino acids 235 to 242; first sequence common to Clades 2 and 3); His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Trp242: "HETMEXLW" (SEQ ID NO: 147) motif (motif 3 of amino acids 235 to 242; second sequence common to Clades 2 and 3); His235-Gln236-Thr237-Asn238-Asp239-X240-Leu241-Phe242: "HETNDXLF" (SEQ ID NO: 148) motif (motif 4 of amino acids 235 to 242; first sequence common to Clade 4); or His235-Gln236-Thr237-Asn238-Asn239-X240-Leu241-Phe242: "HETNNXLF" (SEQ ID NO: 149) motif (motif 5 of amino acids 235 to 242; second sequence common to Clade 4).

[0075] Note that since there may be a small number of insertions or deletions (e.g., five or fewer) of amino acid residues in the amino acid sequence, the positions of the amino acid residues are numbered based on the reference sequence, SEQ ID NO: 2.

[0076] The present invention has been made based on the above findings. The present invention will be described in more detail below.

[0077] 2. L-lysine 5-hydroxylase of the present invention, etc. The L-lysine 5-hydroxylase of the present invention is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, or a polypeptide having a homologue of the amino acid sequence, and has L-lysine 5-hydroxylating activity.

[0078] The L-lysine 5-hydroxylase of the present invention is, for example, a polypeptide belonging to the above Clade 1, 2, 3, 4, or 10, and has the activity of hydroxylating L-lysine 5-position.

[0079] Specifically, the L-lysine 5-hydroxylase of the present invention preferably has a polypeptide shown in the following (A), (B), (C), (D), (E), or (F): (A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134. (B) A polypeptide having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids are deleted, substituted, and / or added, and having L-lysine 5-hydroxylation activity. (C) A polypeptide having an amino acid sequence having 55% or more identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and having L-lysine 5-hydroxylase activity. (D) A polypeptide having an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having L-lysine 5-hydroxylase activity. (E) a polypeptide having an amino acid sequence having 58% or more identity to the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 and having L-lysine 5-hydroxylation activity; (F) a motif of a consecutive amino acid sequence "HGWHWDD";and any one of the motifs of a consecutive amino acid sequence selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF"; and a polypeptide having an activity of hydroxylating L-lysine at the 5-position;

[0080] Polypeptide (A) has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 (hereinafter, sometimes referred to as "amino acid sequence (a) group"). The polypeptide having the amino acid sequence (a) group has an activity of hydroxylating L-lysine at the 5-position, and can hydroxylate L-lysine at the 5-position to produce 5-hydroxy-L-lysine.

[0081] The polypeptide (A) is a polypeptide belonging to Class 1, 2, 3, 4 or 10 and has an activity of hydroxylating L-lysine at the 5-position.

[0082] Among the polypeptides having the amino acid sequence of group (a), polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 22, 26, 28, 30, 34, 36, 40, 44, 46, 52, 74, 82, 84, 90, 98, 100, or 104 are preferred, and polypeptides having the amino acid sequences shown in SEQ ID NO: 8, 10, 16, 90, and 104 are more preferred, because they have high L-lysine 5-hydroxylation activity and a high yield of 5-hydroxy-L-lysine.

[0083] Furthermore, among the polypeptides having the amino acid sequence of group (a), polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 are capable of S-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine. These polypeptides belong to Clade 1. Among these, polypeptides having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, or 22 are preferred, and polypeptides having the amino acid sequence shown in SEQ ID NO: 8, 10, or 16 are more preferred, because they have a high activity of hydroxylating the 5-position of L-lysine and a high yield of (2S,5S)-5-hydroxy-L-lysine.

[0084] Furthermore, among polypeptides having the amino acid sequence of group (a), polypeptides having the amino acid sequence set forth in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 are capable of R-selective hydroxylation at the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine. These polypeptides belong to Class 2, 3, 4, or 10. Among these, polypeptides having the amino acid sequence shown in SEQ ID NO: 26, 28, 30, 34, 36, 40, 44, 46, 52, 74, 82, 84, 90, 98, 100, or 104 are preferred, and polypeptides having the amino acid sequence shown in SEQ ID NO: 90 or 104 are more preferred, because they have a high L-lysine 5-hydroxylation activity and a high yield of (2S,5R)-5-hydroxy-L-lysine.

[0085] Polypeptide (B), (C), (D), (E) or (F) is a polypeptide having a homologue of the amino acid sequence of group (a) and having an activity of hydroxylating the 5-position of L-lysine.

[0086] Polypeptide (B) is a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence group (a), and having L-lysine 5-hydroxylating activity. In the case of substitution or addition, conservative mutations in which one or more amino acids have been conservatively substituted or added are preferred. Here, "one or more amino acids" generally refers to 1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 2, and most preferably 1 amino acid.

[0087] In one embodiment, the polypeptide (B) is a polypeptide belonging to Clade 1, 2, 3, 4 or 10, and has an activity of hydroxylating L-lysine at the 5-position.

[0088] Polypeptide (C) is a polypeptide having a homolog of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among homologs of amino acid sequence (a) group, and having L-lysine 5-hydroxylation activity. In one embodiment, polypeptide (C) belongs to Clade 1.

[0089] Homologs of the amino acid sequences have a sequence identity of 55% or more with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

[0090] Polypeptide (C) is capable of S-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine.

[0091] Polypeptide (D) is a polypeptide having a homolog of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 among homologs of amino acid sequence (a) group, and having L-lysine 5-hydroxylation activity. In one embodiment, polypeptide (D) belongs to Clade 2 or 3.

[0092] Homologs of the amino acid sequences have a sequence identity of 50% or more to the full-length amino acid sequences set forth in SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

[0093] Polypeptide (D) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine.

[0094] Polypeptide (E) is a polypeptide having a homolog of SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 among homologs of amino acid sequence (a) group, and retaining L-lysine 5-hydroxylation activity. In one embodiment, polypeptide (E) belongs to Clade 4.

[0095] Homologs of the amino acid sequences have a sequence identity of 58% or more with the full-length amino acid sequence set forth in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134. Among these, from the viewpoint of L-lysine 5-hydroxylation activity, those having a sequence identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more are preferred.

[0096] Polypeptide (E) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine.

[0097] Polypeptide (F) is a polypeptide that is a homolog of the amino acid sequence (a) group and has a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," and that retains L-lysine 5-hydroxylation activity. More specifically, polypeptide (F) has a consecutive amino acid sequence motif "HGWHWDD" and any one consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF."

[0098] Specific motifs of the consecutive amino acid sequences are as follows: Since there may be a small number of insertions or deletions of amino acid residues (for example, 5 or less) in the amino acid sequences, the positions of the amino acid residues are numbered based on the reference sequence, SEQ ID NO: 2. "HGWHWDD" motif: His136-Gly137-Trp138-His139-Trp140-Asp141-Asp142 "HETMEXLF" motif: His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Phe242 "HETMDXLW" motif: His235-Gln236-Thr237-Met238-Asp239-X240-Leu241-Trp242 "HETMEXLW" motif: His235-Gln236-Thr237-Met238-Gln239-X240-Leu241-Trp242 "HETNDXLF" motif: His235-Gln236-Thr237-Asn238-Asp239-X240-Leu241-Phe242 "HETNNXLF" motif: His235-Gln236-Thr237-Asn238-Asn239-X240-Leu241-Phe242

[0099] These consecutive amino acid sequence motifs, "HGWHWDD," "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," play important roles in expressing L-lysine 5-hydroxylation activity. Furthermore, "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF" play important roles in determining the optical selectivity of L-lysine 5-hydroxylation activity. In particular, "HETMEXLF" plays an important role in expressing S-selective L-lysine 5-hydroxylation activity, while "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF" play important roles in expressing R-selective L-lysine 5-hydroxylation activity.

[0100] Among the polypeptides (F), the following polypeptides (F-1) to (F-3) are preferred.

[0101] Polypeptide (F-1) is a polypeptide that is a homolog of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20, or 22 among homologs of the amino acid sequence (a) group, has an amino acid sequence containing consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF," and has L-lysine 5-hydroxylating activity.

[0102] Polypeptide (F-1) is capable of S-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5S)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-1) belongs to Clade 1.

[0103] Polypeptide (F-2-1) is a homolog of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 among homologs of the amino acid sequence (a) group, and has an amino acid sequence having consecutive amino acid sequence motifs "HGWHWDD" and either "HETMDXLW" or "HETMEXLW", and is a polypeptide having L-lysine 5-hydroxylation activity.

[0104] Polypeptide (F-2-1) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-2-1) belongs to Class 2 or 3.

[0105] Polypeptide (F-2-2) is a homolog of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 among homologs of the amino acid sequence (a) group, and has an amino acid sequence having consecutive amino acid sequence motifs "HGWHWDD" and either "HETNDXLF" or "HETNNXLF," and is a polypeptide having L-lysine 5-hydroxylation activity.

[0106] Polypeptide (F-2-2) is capable of R-selectively hydroxylating the 5-position of L-lysine and can be suitably used for producing (2S,5R)-5-hydroxy-L-lysine. In one embodiment, polypeptide (F-2-2) belongs to Class 4.

[0107] Furthermore, a comparison of overall identity (%) among the L-lysine 5-hydroxylase enzymes exemplified herein showed that even enzymes that share the signature motif but have low identity with each other (for example, an enzyme with only 31% identity to SEQ ID NO: 2) have significant L-lysine 5-hydroxylation activity.

[0108] It should be noted that none of the amino acid sequences in the amino acid sequence (a) group have been reported to have actually been confirmed to exist, such as by being isolated as a protein, and their function as proteins has been completely unknown.

[0109] Furthermore, the L-lysine 5-hydroxylase of the present invention has the following two conserved motifs 1) and 2), which are signature motifs associated with enzymes that primarily catalyze hydroxylation reactions among 2-oxoglutarate-dependent dioxygenases, and is considered to be classified as a 2-oxoglutarate-dependent dioxygenase belonging to the E.C. number 1.14.11. In the following 1) and 2), the positions of amino acid residues are numbered based on the amino acid sequence of the AzpK2 protein (SEQ ID NO: 2). 1) His139-X140-Asp141: "HXD" motif 2) Tyr197-Arg216: "Y-R" motif

[0110] The L-lysine 5-hydroxylase of the present invention can be obtained, for example, by purification from the source organisms shown in Table 1. Alternatively, the L-lysine 5-hydroxylase can be obtained by cloning a DNA encoding the L-lysine 5-hydroxylase by known methods such as PCR or hybridization or by using a chemically synthesized nucleic acid, and then expressing the cloned DNA in an appropriate host.

[0111] Examples of DNA encoding an L-lysine 5-hydroxylase having an amino acid sequence in the amino acid sequence (a) group include DNAs having the nucleotide sequences shown in Table 2. Furthermore, a homologue of the DNA having the nucleotide sequence shown in Table 2 may be used as long as it encodes the L-lysine 5-hydroxylase of the present invention.

[0112] Examples of DNA encoding the L-lysine 5-hydroxylase of the present invention include the DNAs shown in (G), (H), (I), (J), (K) and (L) below.

[0113] (G) is a DNA encoding an L-lysine 5-hydroxylase having the amino acid sequence (a) group, and having the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 (hereinafter, sometimes referred to as "base sequence (g) group").

[0114] (H) to (L) are DNAs which are homologues of DNAs having the base sequence group (g) and encode polypeptides having the activity of hydroxylating the 5-position of L-lysine.

[0115] (H) is a DNA having a base sequence in the base sequence group (g) in which one or more bases have been substituted, deleted, and / or added, and encoding a polypeptide having L-lysine 5-hydroxylating activity. In the case of substitution or addition, a conservative mutation in which one or more bases have been conservatively substituted or added is preferred. The "one or more bases" referred to here generally means 1 to 150, preferably 1 to 60, more preferably 1 to 30, even more preferably 1 to 15, particularly preferably 1 to 5, even more particularly preferably 1 to 3, even more particularly preferably 1 to 2, and most preferably 1.

[0116] (I) is a DNA that is a homolog of a DNA having the base sequence (g) group, and that has 55% or more identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encodes a polypeptide having L-lysine 5-hydroxylating activity. "55% or more identity" as used herein generally means 55% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more identity.

[0117] (J) is a DNA that is a homolog of a DNA having the base sequence (g) group, and that has 50% or more identity to a polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and encodes a polypeptide having L-lysine 5-hydroxylating activity. "50% or more identity" as used herein generally means 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more identity.

[0118] (K) is a DNA that has 58% or more identity with the amino acid sequence encoded by any of DNAs having the nucleotide sequence set forth in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, among homologs of DNAs having the nucleotide sequence of the nucleotide sequence (g) group, and encodes a polypeptide having L-lysine 5-hydroxylating activity. "58% or more identity" as used herein generally means 58% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, even more particularly preferably 97% or more, even more particularly preferably 98% or more, and most preferably 99% or more identity.

[0119] (L) is a DNA homologue of a DNA having the base sequence (g) group, which has a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMEXLF," "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," and encodes a polypeptide having L-lysine 5-hydroxylating activity.

[0120] Those skilled in the art can obtain a homologue of the above-mentioned DNA by introducing substitution, deletion, insertion, and / or additional mutations into the DNA of the base sequence (g) group 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.

[0121] Furthermore, it is also possible to obtain amino acid information of L-lysine 5-hydroxylase or information on the nucleotide sequence of the DNA encoding it by performing a homology search in a database such as the DNA Databank of JAPAN (DDBJ) based on the amino acid sequence of the amino acid sequence (a) group or a part thereof or the nucleotide sequence of the nucleotide sequence (g) group or a part thereof.

[0122] In the method for producing 5-hydroxy-L-lysine of the present invention described below, L-lysine 5-hydroxylase may be used directly in the reaction, or cells containing L-lysine 5-hydroxylase, a preparation of the cells, or a culture medium obtained by culturing the cells may be used.

[0123] As the cells containing L-lysine 5-hydroxylase, cells of a microorganism or the like that originally contain L-lysine 5-hydroxylase may be used, but in terms of ease of availability, it is preferable to use cells of a microorganism or the like that have been transformed with DNA encoding L-lysine 5-hydroxylase. Here, the cells may be live or dead, and for example, resting cells or the like can be preferably used in terms of ease of availability.

[0124] Furthermore, examples of cell preparations containing L-lysine 5-hydroxylase include preparations that contain L-lysine 5-hydroxylase and have the enzyme activity, such as treated cells obtained by treating the cells with an organic solvent such as acetone, dimethyl sulfoxide, or toluene, or a surfactant, freeze-drying the cells, or physically or enzymatically disrupting the cells; crude or purified enzyme fractions obtained from cells; and treated or obtained cells immobilized on a carrier such as polyacrylamide gel or carrageenan gel.

[0125] The culture medium obtained by culturing cells containing L-lysine 5-hydroxylase is a culture medium that contains L-lysine 5-hydroxylase and has the enzyme activity, and may be, for example, a suspension of the cells and a liquid medium, or, when the cells are secretory expression cells, a supernatant obtained by removing the cells by centrifugation or the like, or a concentrate thereof.

[0126] Methods for producing cells (transformants) of microorganisms or the like transformed with DNA encoding L-lysine 5-hydroxylase include, for example, a method of introducing DNA encoding L-lysine 5-hydroxylase into a plasmid vector, phage vector, or viral vector that is stable in host cells of microorganisms or the like, and then introducing the constructed expression vector into the host cells, and a method of directly introducing the DNA into the host genome and transcribing and translating the genetic information. In this case, it is preferable to link an appropriate promoter upstream of the 5'-end of the DNA in the host, and it is more preferable to link a terminator downstream of the 3'-end. Such promoters and terminators are not particularly limited as long as they are known to function in cells used as the host. For example, vectors, promoters, and terminators that can be used in host cells, such as those described in "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan," can be used.

[0127] Specifically, a transformant having DNA encoding L-lysine 5-hydroxylase introduced therein can be obtained by transforming a host cell with an L-lysine hydroxylase expression vector obtained by expressibly inserting DNA encoding L-lysine 5-hydroxylase into a known expression vector. Alternatively, a transformant can be obtained by expressibly incorporating DNA encoding L-lysine 5-hydroxylase into the chromosomal DNA of a host cell by a technique such as homologous recombination.

[0128] The host cells (microorganisms) to be transformed to express L-lysine 5-hydroxylase are not particularly limited as long as they do not adversely affect the L-lysine hydroxylation reaction, and specific examples include the following microorganisms: Bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, etc., for which host vector systems have been established; and Actinomycetes belonging to the genera Rhodococcus, Streptomyces, etc., for which host vector systems have been established. 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. Molds for which host-vector systems have been established, such as those belonging to the genera Neurospora, Aspergillus, Cephalosporium, and Trichoderma.

[0129] The procedures for preparing transformants, the construction of recombinant vectors compatible with 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, bioengineering, and genetic engineering (for example, the methods described in Molecular Cloning).

[0130] 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.

[0131] 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, etc. Examples of terminators include those derived from trpA, phage, and rrnB ribosomal RNA.

[0132] 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.

[0133] For the genus Pseudomonas, examples of vectors include general host vector systems established for Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the degradation 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, 273-282 (1983)).

[0134] In addition to the above, host-vector systems have been established for various microorganisms, and these can be used as appropriate.

[0135] In addition to microorganisms, various host-vector systems have been established in plants and animals. For example, systems for expressing large amounts of heterologous proteins in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)) and in plants such as rapeseed, corn, and potato, as well as cell-free protein synthesis systems such as Escherichia coli cell-free extracts and wheat germ can be suitably used.

[0136] Among the L-lysine 5-hydroxylases of the present invention, those containing the polypeptide shown in (A-1), (B-1), (C-1), or (F-1) below are S-selective L-lysine 5-hydroxylases. (A-1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22. (B-1) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (C-1) A polypeptide having an amino acid sequence having 55% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and retaining S-selective L-lysine 5-hydroxylation activity.

[0137] These S-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-1), (H-1), (I-1), or (L-1) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and then expressing the cloned DNA in an appropriate host. (G-1) DNA containing the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21. (H-1) DNA having a base sequence in which one or more bases are substituted, deleted and / or added in the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21, and encoding a polypeptide having L-lysine 5-hydroxylation activity. (I-1) DNA having 55% or more sequence identity with the polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21, and encoding a polypeptide having S-selective L-lysine 5-hydroxylation activity. (L-1) DNA having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and encoding a polypeptide having S-selective L-lysine 5-hydroxylation activity.

[0138] Furthermore, among the L-lysine 5-hydroxylases of the present invention, those containing the polypeptides shown in the following (A-2), (B-2), (D-2), (E-2), or (F-2) are R-selective L-lysine 5-hydroxylases: (A-2) A polypeptide having the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134. (B-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids have been deleted, substituted, and / or added, and having R-selective L-lysine 5-hydroxylation activity. (D-2) A polypeptide having an amino acid sequence having 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity. (E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, and having R-selective L-lysine 5-hydroxylation activity. (F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity.

[0139] These R-selective L-lysine 5-hydroxylases can be obtained, for example, by cloning the DNA shown in (G-2), (H-2), (J-2), (K-2), or (L-2) below by a known method such as PCR or hybridization or by using a chemically synthesized nucleic acid, and then expressing the cloned DNA in an appropriate host.(G-2) DNA containing the base sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133 (H-2) DNA encoding a polypeptide having an R-selective L-lysine 5-hydroxylating activity, comprising a nucleotide sequence represented by SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, in which one or more nucleotides have been substituted, deleted, and / or added. (J-2) DNA encoding a polypeptide having 50% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and having R-selective L-lysine 5-hydroxylation activity. (K-2) DNA encoding a polypeptide having 58% or more sequence identity with a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and having R-selective L-lysine 5-hydroxylation activity. (L-2) DNA encoding a polypeptide having a consecutive amino acid sequence motif of "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW," "HETMEXLW," "HETNDXLF," and "HETNNXLF," and having R-selective L-lysine 5-hydroxylation activity.

[0140] 3. Enzyme Composition The enzyme composition of the present invention contains the L-lysine 5-hydroxylase of the present invention, or cells containing the same, a preparation of the cells, or a culture medium obtained by culturing the cells (hereinafter, these may be collectively referred to as the "L-lysine 5-hydroxylase of the present invention, etc."), and has the L-lysine 5-hydroxylase activity. When used as a catalyst, the enzyme composition of the present invention can produce 5-hydroxy-L-lysine from L-lysine in high yield and at low cost. Furthermore, the enzyme composition of the present invention has high regioselectivity and stereoselectivity when hydroxylating L-lysine, and therefore can efficiently produce the desired optical isomer of 5-hydroxy-L-lysine.

[0141] The enzyme composition of the present invention may contain, in addition to the active ingredient, the L-lysine 5-hydroxylase of the present invention, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, and the like. Examples of excipients that can be used include lactose, sorbitol, D-mannitol, and sucrose. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of stabilizers that can be used include propylene glycol and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. The content of the L-lysine 5-hydroxylase in the enzyme composition of the present invention is appropriately set within a range that allows the effects of the L-lysine 5-hydroxylase to be exerted. For example, the content is appropriately set so as to achieve the amount used in the method for producing 5-hydroxy-L-lysine of the present invention described below. For example, the enzyme composition may be blended in an amount of 0.01 to 100% by weight, 0.1 to 10% by weight, or 1 to 50% by weight of the total enzyme composition. The enzyme composition of the present invention can be produced by a commonly used formulation method.

[0142] 4. Method for Producing 5-hydroxy-L-lysine Using L-lysine 5-Hydroxylase The method for producing 5-hydroxy-L-lysine of the present invention involves contacting L-lysine with the L-lysine 5-hydroxylase of the present invention, or a cell containing it, a preparation of the cell, or a culture medium obtained by culturing the cell, thereby producing 5-hydroxy-L-lysine by the reaction of formula (I) below:

[0143]

[0144] The method is characterized in that it produces 5-hydroxy-L-lysine represented by the formula:

[0145] The formula (I) includes the following formula (II) in which the hydroxyl group at the 5-position is in the S-configuration:

[0146]

[0147] Or, a compound represented by the following formula (III) in which the hydroxyl group at the 5-position is in the R configuration:

[0148]

[0149] It is preferable that either one of the above is used.

[0150] The compound represented by formula (II) is (2S,5S)-5-hydroxy-L-lysine, and the compound represented by formula (III) is (2S,5R)-5-hydroxy-L-lysine.

[0151] In the production method of the present invention, when the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (A) and / or (B), 5-hydroxy-L-lysine represented by formula (I) can be produced. When the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (C), (2S,5S)-5-hydroxy-L-lysine represented by formula (II) can be produced. When the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (D) or (E), (2S,5R)-5-hydroxy-L-lysine represented by formula (III) can be produced. When the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (F), 5-hydroxy-L-lysine represented by formula (I) can be produced; when the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptide (F-1), (2S,5S)-5-hydroxy-L-lysine represented by formula (II); and when the L-lysine 5-hydroxylase or the like of the present invention contains the polypeptides (F-2) and / or (F-3), (2S,5R)-5-hydroxy-L-lysine represented by formula (III).

[0152] In the production method of the present invention, multiple types of L-lysine 5-hydroxylase etc. may be used in combination. In this case, it is preferable to use L-lysine 5-hydroxylase etc. having the same stereoselectivity in combination.

[0153] The method for contacting L-lysine with the L-lysine 5-hydroxylase or the like of the present invention is not particularly limited as long as it allows contact between the two. However, it is generally preferable to carry out the contact in a liquid such as an aqueous medium or a mixture of an aqueous medium and an organic solvent. Furthermore, in the case of a mixture of an aqueous medium and an organic solvent, it is preferable that the ratio of the aqueous medium is large. Examples of aqueous media include water and known buffers such as Good's buffer, phosphate buffer, Tris buffer, and borate buffer. Examples of organic solvents that can be used include those that have high solubility for the reaction substrate, L-lysine, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, and dimethyl sulfoxide. Furthermore, examples of organic solvents that can be used include ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and methyl tert-butyl ether, which are effective for removing reaction by-products.

[0154] Examples of methods for contacting L-lysine with the L-lysine 5-hydroxylase or the like of the present invention to cause a reaction include adding the reaction substrate L-lysine to a liquid containing the L-lysine 5-hydroxylase or the like of the present invention, or adding the L-lysine 5-hydroxylase or the like of the present invention to a liquid containing L-lysine. From the viewpoints of reducing the influence of any inhibitory action on the enzyme, increasing the accumulated concentration of the target product, and reducing by-products, it is preferable to add the reaction substrate L-lysine to a liquid containing the L-lysine 5-hydroxylase or the like of the present invention.

[0155] When L-lysine comes into contact with the L-lysine 5-hydroxylase or the like of the present invention and reacts with it, L-lysine is hydroxylated to produce 5-hydroxy-L-lysine.

[0156] The amount of the L-lysine 5-hydroxylase or the like of the present invention used is not particularly limited, as long as it is an amount that can hydroxylate L-lysine to produce 5-hydroxy-L-lysine. For example, when cells containing L-lysine 5-hydroxylase are used, the cells can be used so that the concentration of the cells in the reaction system (e.g., in the reaction solution) is typically 0.1 g / L to 50 g / L, and preferably 1 g / L to 20 g / L, based on wet cell weight. Furthermore, when a preparation of the cells or a culture solution obtained by culturing the cells is used, the specific activity of the L-lysine 5-hydroxylase used is determined, and an amount can be used such that the concentration of the cells in the reaction system (e.g., in the reaction solution) is typically 0.1 g / L to 50 g / L, and preferably 1 g / L to 20 g / L, based on wet cell weight.

[0157] L-lysine, which is a reaction substrate, can be used so that the substrate concentration in the reaction system (e.g., in the reaction solution) is generally in the range of 0.01 mM to 200 mM, preferably 10 mM to 200 mM, more preferably 20 mM to 200 mM, and particularly preferably 50 mM to 200 mM.

[0158] As the substrate L-lysine, commercially available lysine can be used, but lysine produced by a biological method using enzymes, bacteria, etc. can also be used.

[0159] As the reaction substrate, L-lysine is preferably used as it is, but compounds such as L-lysine hydrochloride that become L-lysine in the reaction system (for example, in the reaction solution) can also be used.

[0160] When adding L-lysine as a reaction substrate to a liquid containing the L-lysine 5-hydroxylase or the like of the present invention, L-lysine may be added all at once at the start of the reaction. However, from the viewpoints of reducing the influence of any inhibitory action on the enzyme, increasing the accumulated concentration of the target product, reducing by-products, etc., it is desirable to add L-lysine continuously or intermittently.

[0161] Furthermore, the contact (reaction) of L-lysine with the L-lysine 5-hydroxylase of the present invention is preferably carried out aerobically in the presence of 2-oxoglutaric acid and divalent iron ions.

[0162] 2-oxoglutaric acid can usually be used in an amount equimolar or more, preferably equimolar to 2-fold molar, more preferably equimolar to 1.8-fold molar, even more preferably equimolar to 1.5-fold molar, and particularly preferably equimolar to 1.2-fold molar, relative to the amount of L-lysine used as a reaction substrate.

[0163] 2-oxoglutaric acid may be added all at once at the start of the reaction. However, from the viewpoints of reducing the influence of any inhibitory action on the enzyme, increasing the accumulated concentration of the target product, reducing by-products, etc., it is desirable to add it continuously or intermittently.

[0164] Instead of 2-oxoglutaric acid, an inexpensive compound such as glucose that can be metabolized by the host can be used and metabolized by the host, and the 2-oxoglutaric acid produced in the metabolic process can be used in the reaction.

[0165] Alternatively, instead of 2-oxoglutaric acid, it is possible to generate 2-oxoglutaric acid from an inexpensive compound using a plurality of enzymes and use the 2-oxoglutaric acid in the reaction.

[0166] Divalent iron ions can be used at a concentration in the reaction system (e.g., in the reaction solution) of usually 0.01 mM to 100 mM, preferably 0.1 mM to 10 mM. Divalent iron ions are preferably added all at once at the start of the reaction as iron sulfate or the like, but if the divalent iron ions added during the reaction are oxidized to trivalent iron or form a precipitate and are reduced, it is also effective to add additional iron ions.

[0167] Furthermore, when the L-lysine hydroxylase or the like of the present invention already contains a sufficient amount of divalent iron ions, it is not necessary to add divalent iron ions.

[0168] The contact (reaction) of L-lysine with the L-lysine 5-hydroxylase or the like of the present invention can usually be carried out under a desired pressure that does not adversely affect the reaction, and at a reaction temperature of usually 4°C to 60°C, preferably 10°C to 45°C, and more preferably 15°C to 30°C. The contact (reaction) can usually be carried out under conditions of pH 3 to 11, preferably pH 5 to 8. The contact (reaction) time is not particularly limited, as long as it is a time sufficient for L-lysine to be hydroxylated to produce the desired 5-hydroxy-L-lysine, and is usually 30 minutes to 72 hours, preferably 1 hour to 24 hours.

[0169] The produced 5-hydroxy-L-lysine can be purified, if necessary, by separating 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 methods known to those skilled in the art, such as extraction with an organic solvent such as 1-butanol or tert-butanol, distillation, column chromatography using an ion exchange resin or silica gel, or crystallization at the isoelectric point or crystallization with monohydrochloride, dihydrochloride, calcium salt, or the like.

[0170] Alternatively, the resulting 5-hydroxy-L-lysine can be purified by introducing a water-insoluble derivative / protecting group into the amino group, followed by extraction with an organic solvent and crystallization. Examples of the organic solvent that can be used include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, and methyl tert-butyl ether.

[0171] In the present invention, (2S,5S)-5-hydroxy-L-lysine can be produced by contacting L-lysine with a polypeptide shown in (A-1), (B-1), (C-1), or (F-1) below, or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells. (A-1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22. (B-1) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (C-1) A polypeptide having an amino acid sequence having 55% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity. (F-1) A polypeptide having consecutive amino acid sequence motifs "HGWHWDD" and "HETMEXLF", and retaining S-selective L-lysine 5-hydroxylation activity.

[0172] Furthermore, in the present invention, (2S,5R)-5-hydroxy-L-lysine can be produced by contacting L-lysine with a polypeptide shown in (A-2), (B-2), (D-2), (E-2), or (F-2) below, or a cell containing the polypeptide, a preparation of the cells, or a culture medium obtained by culturing the cells.(A-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134 (B-2) A polypeptide having an amino acid sequence represented by SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, in which one or more amino acids have been deleted, substituted, and / or added, and having R-selective L-lysine 5-hydroxylation activity. (D-2) A polypeptide having an amino acid sequence having 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110, and having R-selective L-lysine 5-hydroxylation activity. (E-2) A polypeptide having an amino acid sequence having 58% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, or 134, and having R-selective L-lysine 5-hydroxylation activity. (F-2) A polypeptide having a consecutive amino acid sequence motif "HGWHWDD" and any consecutive amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF" and "HETNNXLF", and having R-selective L-lysine 5-hydroxylation activity.

[0173] As described above, the method for producing 5-hydroxy-L-lysine of the present invention can produce the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine by using the appropriate L-lysine 5-hydroxylase.

[0174] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0175] Example 1 (Cloning of azpK2 Gene) Actinosynnema mirum JCM3225, purchased from the Microorganism Materials Division of the RIKEN BioResource Research Center (RIKEN BRC) (hereinafter referred to as "JCM"), was used to obtain genomic DNA according to a standard genomic DNA extraction method using lysozyme, SDS, chloroform, and isopropanol precipitation. Using this extracted genomic DNA as a template, a PCR reaction was performed using the primers shown in SEQ ID NOs: 150 and 151 to amplify the azpK2 gene. PrimeStar HS DNA polymerase (manufactured by Takara Bio Inc.) was used in the PCR reaction to obtain an amplification product of the target azpK2 gene. The composition of the reaction solution and the reaction cycle are as follows:

[0176] PCR reaction solution composition Sterile water: 8.6 μL 5 mol / L Betaine: 4.0 μL 5×PrimeSTAR (registered trademark) Buffer: 4.0 μL 2.5 mM dNTP mix: 1.6 μL AzpK2_F (100 μM): 0.6 μL AzpK2_R (100μM): 0.6μL Genomic DNA (2.5-25μg / μL): 0.4μL PrimeStar (registered trademark) HS DNA polymerase: 0.2μL

[0177]

[0178] The pCold I vector (manufactured by Takara Bio Inc.) treated with restriction enzymes NdeI and XhoI (manufactured by Takara Bio Inc.) and the PCR product were ligated using an In-Fusion® HD Cloning Kit (manufactured by Takara Bio Inc.) according to standard methods. The resulting product was then transformed into E. coli JM109 strain (manufactured by Takara Bio Inc.) according to standard methods. Plasmid DNA was extracted from the resulting colonies by miniprep according to standard methods to obtain the pCold I-AzpK2 vector.

[0179] Example 2 (Cloning of azpJ gene) Genomic DNA was obtained using the actinomycete Streptacidiphilus griseoplanus JCM4300 purchased from JCM in the same manner as in Example 1, and the primers shown in SEQ ID NOs: 152 and 153 were used in the same manner as in Example 1 to obtain the pCold I-AzpJ vector for expressing the AzpJ protein shown in SEQ ID NO: 140 (the base sequence of DNA encoding the amino acid sequence of this protein is SEQ ID NO: 139).

[0180] Example 3 (Preparation of Expression Vectors for Pp_azpK2 Gene, azpK2 Homolog Gene, and Pp_azpJ Gene) The Pp_azpK2 gene, azpK2 homolog gene, and Pp_azpJ gene encoding the Pp_azpJ protein shown in Table 3 below, were optimized to have a codon frequency suitable for expression in E. coli, and synthetic genes in which TCGAAGGTAGGCATATG (SEQ ID NO: 154) and TAACTCGAGGGATCCGAAT (SEQ ID NO: 155) were added upstream and downstream of the gene, respectively, as sequences homologous to the pCold I vector, were added to the nucleic acid sequence of each SEQ ID NO. These synthetic genes were purchased from Thermo Fisher Scientific.

[0181] The pCold I vector (manufactured by Takara Bio Inc.) was treated with the restriction enzymes NdeI and XhoI (manufactured by Takara Bio Inc.) and ligated together with the synthetic gene according to standard procedures using an In-Fusion (registered trademark) HD ​​Cloning Kit (manufactured by Takara Bio Inc.). Subsequently, Escherichia coli JM109 strain was transformed according to standard procedures, and plasmid DNA was extracted from the resulting colonies using the miniprep method to prepare the expression vectors listed in Table 3 below.

[0182]

[0183] Example 4 (Preparation of Aqueous Recombinant Protein Solution-1) Using the expression vectors obtained in Examples 1 to 3, E. coli BL21 (DE3) strain (Merck) was transformed according to a standard method. The resulting colonies were inoculated into 2 mL of LB medium (hereinafter referred to as "LB (Amp50) medium") containing ampicillin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) at a final concentration of 50 μg / mL, and cultured overnight with shaking at 37°C.

[0184] The resulting culture was inoculated at 1% volume into Terrific Broth (hereinafter referred to as "TB (Amp50) medium") containing a final concentration of 50 μg / mL sodium ampicillin, and cultured with shaking (150 rpm) at 37°C. When the absorbance (OD600nm) reached 0.5 to 0.8, the medium was cooled to 15°C, and IPTG was added to a final concentration of 20 μM or 50 μM for induction. After culture with shaking (150 rpm) at 15°C for 24 hours, the cells were collected from the culture by centrifugation (20,000 × g, 10 minutes, 4°C). The cells were resuspended in lysis buffer (20 mM HEPES, 10% W / V glycerol, 200 mM sodium chloride (hereinafter referred to as "NaCl"), pH 8.0) and sonicated using a Q500 Sonicator (manufactured by QSonica). The resulting solution was then centrifuged (20,000 x g, 60 minutes, 4°C) to obtain an aqueous solution containing the recombinant protein. His60 Ni Superflow Resin (manufactured by Takara Bio Inc.) was added to the resulting solution and stirred to allow the recombinant protein to bind, which was then transferred to a column.

[0185] Elution buffer (20 mM HEPES, 10% glycerol, 200 mM NaCl, 500 mM imidazole, pH 8.0) and lysis buffer were mixed to prepare aqueous solutions with imidazole concentrations of 20 mM, 50 mM, 100 mM, and 250 mM, respectively. The recombinant protein was eluted from the resin in the column by adding aqueous solutions with increasing imidazole concentrations to the column in stages.

[0186] The elution fractions at each imidazole concentration were confirmed for the presence or absence of recombinant protein by polyacrylamide gel electrophoresis (SDS-PAGE), and the elution fractions in which the presence was confirmed were concentrated and desalted by ultrafiltration (5000 x g, 4 °C) using Amicon Ultra-15 Centrifugal Filters (10 kDa) (Merck). The protein concentration of the resulting aqueous protein solution was measured using a NanoDrop (registered trademark) microspectrophotometer (Thermo Fisher Scientific) and then frozen and stored. The concentration of the aqueous protein solution prepared by this method was calculated using the approximate extinction coefficient at A280 nm calculated from the amino acid sequence and the predicted molecular weight of the amino acid.

[0187] Example 5 (Confirmation of Lysine 5-Hydroxylation Activity of AzpK2, Pp_AzpK2, and AzpK2 Homologues) 0.1 mM L-lysine (hereinafter referred to as "L-Lys"), 0.1 mM α-ketoglutaric acid (hereinafter sometimes referred to as "αKG"), 1.0 mM ascorbic acid, 25 μM FeSO ４ , 20 mM HEPES, 10 W / V% glycerol, 200 mM NaCl (pH 8.0), 5.0 μM recombinant protein of AzpK2, Pp_AzpK2, or AzpK2 homolog (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) obtained in Example 4 was used to prepare 100 μL of the reaction solution, and the reaction was carried out at 30 ° C for 1 hour. After completion of the reaction, the reaction was stopped by adding 100 μL of methanol, and the supernatant obtained by centrifugation was analyzed by LC-MS (analysis condition 1). Details of analysis condition 1 are as follows.

[0188]

[0189] As a result of the analysis, a peak at m / z 163.2 was detected at the same retention time (4 min) as 5-hydroxy-L-lysine (Merck) in all cases where aqueous solutions of recombinant proteins of AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homolog 1-1 to 1-9, 2-1 to 2-3, 3-1 to 3-2) were used.

[0190] Furthermore, 50 μL of the reaction solution prepared above was taken and mixed with 50 μL of 1 mM fluorenylmethyloxycarbonyl chloride (hereinafter sometimes referred to as "Fmoc-Cl") dissolved in acetonitrile, and the mixture was reacted at 30°C for 30 minutes. 50 μL of 1 M HCl was added to this mixture to terminate the reaction, and the reaction product was extracted with 150 μL of ethyl acetate. The solvent was removed using a centrifugal evaporator, and the remaining solid was dissolved in 20 μL of methanol to obtain an Fmoc-treated sample in which the amino acid was Fmoc-treated. This Fmoc-treated sample was analyzed by LC-MS (analysis condition 2). Details of analysis condition 2 are as follows.

[0191]

[0192] As a result of the analysis, a peak at m / z 607.2 was detected at the same retention time (8 min) as that of the di-Fmoc-modified 5-hydroxy-L-lysine (Merck & Co.) in all cases where aqueous solutions of recombinant proteins of AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homologs 1-1 to 1-9, 2-1 to 2-3, and 3-1 to 3-2) were used. Therefore, it was found that AzpK2, Pp_AzpK2, and AzpK2 homologs (AzpK2 Homologs 1-1 to 1-9, 2-1 to 2-3, and 3-1 to 3-2) all have L-lysine 5-hydroxylation activity.

[0193] FIG. 3 shows the results of LC-MS detection of the reaction product when AzpK2 was used (detection results of di-Fmoc-modified 5-hydroxy-L-lysine with MS +607.3).

[0194] "Positive control" in Figure 3 is the positive control obtained by analyzing a di-Fmoc-modified 5-hydroxy-L-lysine, and the peak at 8.0 min is the di-Fmoc-modified 5-hydroxy-L-lysine. "No AzpK2" is a negative control obtained by omitting AzpK2 from the total reaction solution composition. "All" is the total reaction solution composition including AzpK2, and the peak of the di-Fmoc-modified 5-hydroxy-L-lysine, the same as that of the positive control, was detected around 8.0 min.

[0195] Example 6 (Measurement of enzymatic parameters of AzpK2, Pp_AzpK2, and AzpK2 homologues using purified enzymes) 0.1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 25 μM FeSO ４ , 20 mM HEPES, 10 W / V% glycerol, 200 mM NaCl (pH 8.0), 1.0 mM NAD ＋ 100 μL of reaction solutions were prepared, each containing a 5.0 μM aqueous solution of the recombinant protein of AzpK2, Pp_AzpK2, or AzpK2 homolog (AzpK2 Homolog 1-1 to 1-3, 2-1 to 2-3, 3-1) obtained in Example 4, a 2.0 μM aqueous solution of the recombinant protein of AzpJ or Pp_AzpJ obtained in Example 4, and L-Lys at concentrations of 0.01 mM, 0.02 mM, 0.1 mM, 0.2 mM, or 0.5 mM. L-Lys was added last after mixing the other components.

[0196] The reaction was initiated by the addition of L-Lys (substrate). The absorbance at a wavelength of 340 nm (the absorbance wavelength of NADH) was measured over time using a SpectraMax M2 (Molecular Devices), and the initial rate of NADH production (initial reaction rate) was determined from the slope of the resulting absorbance change.

[0197] The obtained initial reaction rates were fitted to the Michaelis-Menten equation to calculate the enzymatic parameters Kcat, Km, and Kcat / Km of each protein. The results are shown in Table 4 below.

[0198]

[0199] As is clear from Table 4, Kcat was highest for AzpK2 Homolog 3-1, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 2-2, in that order. Furthermore, Kcat / Km was highest for AzpK2 Homolog 1-2, followed by Pp_AzpK2, AzpK2 Homolog 2-3 and AzpK2 Homolog 1-1, in that order.

[0200] Example 7 (Confirmation of hydroxylation activity of L-lysine 5-hydroxylase on 5-hydroxy-L-lysine) The presence or absence of 5-hydroxylation activity on 5-hydroxy-L-lysine was measured for AzpK2 Homologs 1-1, 1-2 and 1-3.

[0201] 0.1 mM 5-hydroxy-L-lysine, 0.1 mM or 1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 25 μM FeSO ４ 100 μL of a reaction solution prepared to the composition of 20 mM HEPES, 10% glycerol, 200 mM NaCl (pH 8.0), and 5.0 μM of the recombinant protein solution of AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, or AzpK2 Homolog 1-3 obtained in Example 4 was used, and the reaction was carried out at 30° C. for 1 hour. The reaction solution was Fmoc-conjugated in the same manner as in Example 5, and analyzed by HPLC (analysis condition 3). Details of analysis condition 3 are as follows.

[0202]

[0203] The results of HPLC analysis are shown in Figure 4. In Figure 4, for AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3, the upper panel shows the results of the reaction under 1.0 mM αKG conditions, and the lower panel shows the results of the reaction under 0.1 mM αKG conditions. In Figure 4, "5-OHLys-Fmoc" indicates the peak of the Fmoc-modified 5-hydroxy-L-lysine, "5-oxoLys-Fmoc" indicates the peak of the Fmoc-modified 5-oxolysine, and "5-OHLys-diFmoc" indicates the peak of the di-Fmoc-modified 5-hydroxy-L-lysine.

[0204] 4, 5-oxolidine was detected under all conditions for AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3. In addition, the detection peak of 5-oxolidine was larger when α-ketoglutaric acid was added at a concentration of 1 mM than when it was added at a concentration of 0.1 mM.

[0205] This suggests that when 5-hydroxy-L-lysine is obtained from L-Lys using L-lysine 5-hydroxylase, the by-production of 5-oxolidine can be suppressed and 5-hydroxy-L-lysine can be obtained in high yield when the concentration of added α-ketoglutaric acid is lower.

[0206] Reference Example 1 (Method for Confirming the Stereochemistry of the Hydroxy Group of 5-Hydroxy-L-lysine) A reaction solution containing about 1 mM 5-hydroxy-L-lysine was dissolved in 20 mM HEPES buffer, 0.2 mM NAD ＋ and 0.46 mg / mL Pseudomonas putida-derived cyclodeaminase (pH 7.0) are added to prepare 1.0 mL of reaction solution, and the mixture is reacted at 30°C with shaking (200 rpm) for about 4 to 24 hours to obtain a reaction solution containing the reaction product, 5-hydroxy-L-pipecolic acid.

[0207] To 50 μL of the resulting reaction solution, 50 μL of 500 mM boric acid (pH 9.0) and 50 μL of 10 mM Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA) are added in that order, and the mixture is incubated at 40°C for 1 hour. After that, 50 μL of 1 M HCl and 200 μL of acetonitrile are added, and the mixture is filtered through a 0.45 μm filter to obtain FDAA-derivatized 5-hydroxy-L-pipecolic acid.

[0208] The resulting solution containing FDAA-derivatized 5-hydroxy-L-pipecolic acid is analyzed by HPLC (Analysis Condition 4 or Analysis Condition 5) and compared with a previously prepared FDAA-derivatized (2S,5S)-5-hydroxy-L-pipecolic acid or a previously prepared FDAA-derivatized (2S,5R)-5-hydroxy-L-pipecolic acid, thereby determining the absolute configuration of the hydroxy group in the reaction product, 5-hydroxy-L-lysine. Details of Analysis Condition 4 and Analysis Condition 5 are as follows.

[0209]

[0210]

[0211] Example 8 (Confirmation of the Stereochemistry of the Hydroxy Group of 5-Hydroxy-L-Lysine) 20 mM HEPES buffer (pH 7.0), 10 mM L-Lys, 20 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 0.5 mM FeSO ４ 1.0 mL of a reaction solution containing 1.0 mL of the 5-hydroxy-L-lysine solution obtained in Example 4 and 3.3 μM of the AzpK2 or Pp_AzpK2 protein aqueous solution was reacted at 28° C. for 14 hours with shaking at 200 rpm to obtain a 5-hydroxy-L-lysine solution.

[0212] This 5-hydroxy-L-lysine solution was reacted with Pseudomonas putida-derived cyclodeaminase in accordance with Reference Example 1 for 24 hours for the 5-hydroxy-L-lysine solution obtained using AzpK2, or for 4 hours for the 5-hydroxy-L-lysine solution obtained using Pp_AzpK2, to obtain reaction solutions containing 5-hydroxy-L-pipecolic acid for each.

[0213] Furthermore, FDAA-derivatized 5-hydroxy-L-pipecolic acid was obtained from each of the resulting reaction solutions containing 5-hydroxy-L-pipecolic acid in accordance with Reference Example 1. The resulting solutions containing FDAA-derivatized 5-hydroxy-L-pipecolic acid were analyzed under analytical condition 4 for the reaction solution obtained using AzpK2 and under analytical condition 5 for the reaction solution obtained using Pp_AzpK2, and compared with the FDAA-derivatized product of (2S,5S)-5-hydroxy-L-pipecolic acid or FDAA-derivatized product of (2S,5R)-5-hydroxy-L-pipecolic acid that had been prepared in advance.

[0214] As a result, when the 5-hydroxy-L-lysine solution obtained using AzpK2 was reacted with cyclodeaminase, the reaction product was (2S,5S)-5-hydroxy-L-pipecolic acid, and when the 5-hydroxy-L-lysine solution obtained using Pp_AzpK2 was reacted with cyclodeaminase, the reaction product was (2S,5R)-5-hydroxy-L-pipecolic acid.

[0215] This demonstrated that AzpK2 has the activity of S-selectively hydroxylating the 5-position of L-lysine, and that Pp_AzpK2 has the activity of R-selectively hydroxylating the 5-position of L-lysine.

[0216] Example 9 (Confirmation of substrate selectivity of AzpJ and Pp_AzpJ) 0.1 mM L-Lys, 0.1 mM α-ketoglutaric acid, 1.0 mM ascorbic acid, 1.0 mM NAD ＋ , 25 μM FeSO ４100 μL of reaction solution was prepared with 5.0 μM of the recombinant protein of the hydroxylase AzpK2 or Pp_AzpK2 obtained in Example 4, 5.0 μM of the recombinant protein of the oxidase AzpJ or Pp_AzpJ obtained in Example 4, 20 mM HEPES, 10% glycerol, and 200 mM NaCl (pH 8.0). The absorbance at a wavelength of 340 nm, which indicates the absorbance of NADH, was measured for 2 minutes at 30 ° C using a SpectraMax M2 (Molecular Devices). The initial rate of NADH production was calculated from the slope of the absorbance change obtained here. The absorbance of NADH was estimated based on the absorbance data of the NADH reagent (Nacalai Tesque).

[0217] As a result, when AzpK2, an S-selective L-lysine 5-hydroxylase, was used, an increase in absorbance at 340 nm due to increased accumulation of NADH was confirmed only when AzpJ was added, whereas when Pp_AzpK2, an R-selective L-lysine 5-hydroxylase, was used, an increase in absorbance at 340 nm due to increased accumulation of NADH was confirmed only when Pp_AzpJ was added.

[0218] In addition, when the reaction solution was analyzed by HPLC (analysis condition 6), Kawai, S. et al. Angew. Chemint. Int. Ed. Engl. 2021, 60, pp. 10319-10325 reported that the compound obtained by cyclization and dehydration of 5-oxo-L-lysine had a mass peak of m / z 143.2. Details of analysis condition 6 are as follows.

[0219]

[0220] These results demonstrate that AzpJ is an enzyme that selectively oxidizes the hydroxy group in (5S)-hydroxy-L-lysine, and that Pp_AzpJ is an enzyme that selectively oxidizes the hydroxy group in (5R)-hydroxy-L-lysine. Therefore, if an oxidation reaction occurs when AzpJ is used in a reaction solution of lysine 5-hydroxylase, resulting in an increase in absorbance derived from NADH, the lysine 5-hydroxylase can be determined to be an S-selective hydroxylase, and if an oxidation reaction occurs when Pp_AzpJ is used in a reaction solution of lysine 5-hydroxylase, resulting in an increase in absorbance derived from NADH, the lysine 5-hydroxylase can be determined to be an R-selective hydroxylase.

[0221] Example 10 (Confirmation of stereoselectivity of hydroxyl group of AzpK2 homolog) Using the protein aqueous solution of the AzpK2 homolog obtained in Example 4, the increase in absorbance at 340 nm, which indicates the absorbance of NADH, was measured according to the procedure of Example 9 when either AzpJ or Pp_AzpJ was used as the oxidase. The results shown in Table 5 were obtained.

[0222]

[0223] Furthermore, as described above in Example 9, the AzpK2 homolog whose absorbance increased with AzpJ is an S-selective L-lysine 5-hydroxylase, and the AzpK2 homolog whose absorbance increased with Pp_AzpJ is an R-selective L-lysine 5-hydroxylase. Therefore, the stereoselectivity of each hydroxylase (AzpK2 homolog obtained in Example 4) is also shown in Table 5.

[0224] When we investigated whether the absorbance of AzpK2 Homolog4-1 increased when either AzpJ or Pp_AzpJ was used as the oxidase, the increase in absorbance was small in both cases, making it difficult to distinguish between them. Therefore, we attempted to detect Fmoc-modified 5-oxolidine using the same method as in Example 7. The results are shown in Figure 5.

[0225] In Figure 5, "AzpK2 + AzpJ" represents a positive control in which 5-oxolidine was produced using the hydroxylase AzpK2 and the oxidase AzpJ, and "4-1" represents a negative control in which the hydroxylase AzpK2 Homolog 4-1 was added but no oxidase was added. "4-1 + AzpJ" represents the analysis result of a reaction solution containing the hydroxylase AzpK2 Homolog 4-1 and the oxidase AzpJ, in which a 5-oxolidine peak slightly larger than that of the negative control was detected. "4-1 + Pp_AzpJ" represents the analysis result of a reaction solution containing the hydroxylase AzpK2 Homolog 4-1 and the oxidase Pp_AzpJ, in which a 5-oxolidine peak clearly stronger than that of "4-1 + AzpJ." That is, more 5-oxolidine was detected when the oxidase Pp_AzpJ was used than when the oxidase AzpJ was used.

[0226] Therefore, it was found that AzpK2 Homolog4-1 has the activity of hydroxylating both the S- and R-isomers of L-lysine at the 5-position, but that the R-isomer-selective hydroxylation activity is higher.

[0227] Example 11 (Comparison of reactivity of L-lysine 5-hydroxylase) In the method of Example 9, the reaction rate of the oxidation of 5-hydroxy-L-lysine was faster than the hydroxylation of L-Lys by L-lysine 5-hydroxylase. Therefore, the difference in the rate of NADH accumulation when using a method similar to that of Example 9 but changing the hydroxylase represents the difference in the rate of L-lysine hydroxylation by the hydroxylase.

[0228] Using the oxidase AzpJ, the reactivity of each hydroxylase to 1 mM L-Lys was compared in the same manner as in Example 9. The NADH accumulation rate (initial rate) for the first 2 minutes was measured, and the results are shown in Table 6.

[0229]

[0230] As is clear from Table 6, among the S-selective L-lysine 5-hydroxylases, the NADH accumulation rate (initial velocity) was highest for AzpK2 Homolog1-2, followed by AzpK2 Homolog1-9, AzpK2 Homolog1-1, and AzpK2 Homolog1-3 in that order. Therefore, it is believed that the hydroxylation activity of the S-selective L-lysine 5-hydroxylases also follows the same order of increasing velocity. Furthermore, among the R-selective L-lysine 5-hydroxylases, the NADH accumulation rate (initial velocity) was highest for AzpK2 Homolog3-1, followed by Pp_AzpK2, AzpK2 Homolog2-2, and AzpK2 Homolog2-3 in that order. Therefore, it is believed that the hydroxylation activity of the R-selective L-lysine 5-hydroxylases also follows the same order of increasing velocity.

[0231] Example 12 (Evaluation of Activities of AzpK2, Pp_AzpK2, and AzpK2 Homologs Using Genetically Recombinant E. coli - 1) Example 12-1: Preparation of pKW32 Expression Vector For AzpK2, Pp_AzpK2, AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3 shown in Table 7 below, linear DNA fragments with an NdeI site added upstream and an XhoI site added downstream of the gene sequences shown in the respective SEQ ID NOs were purchased from Eurofin Genomics.

[0232]

[0233] The synthetic genes of Pp_AzpK2, AzpK2 Homolog1-1, AzpK2 Homolog1-2, and AzpK2 Homolog1-3 obtained here were subjected to PCR according to standard methods using PrimeStar max DNA polymerase (Takara Bio Inc.) and each primer sequence designed to provide restriction enzyme EcoRI and XbaI sites to each gene shown in Table 8 below, to amplify gene fragments having the restriction enzyme EcoRI and XbaI sites.

[0234]

[0235] In addition, the pKW32 vector (obtained with reference to Japanese Patent No. 5613660) was cleaved with restriction enzymes MunI and XbaI (both manufactured by Takara Bio Inc.), and then dephosphorylated using TSAP (Thermosensitive Alkaline Phosphatase, manufactured by Promega Corp.) to prepare a DNA fragment. The PCR product obtained above was cleaved with restriction enzymes EcoRI and XbaI (manufactured by Takara Bio Inc.) and ligated using a DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.) according to a standard method. Then, the resulting strain was transformed into Escherichia coli JM109 (manufactured by Takara Bio Inc.) according to a standard method. The resulting strain was cultured overnight at 30 ° C. on LB agar medium (hereinafter referred to as LB (Km50) agar medium) containing 50 μg / mL kanamycin sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). From the resulting colonies, expression vectors shown in Table 8 under the names of the constructed vectors were obtained according to standard methods using a QIAprep Spin miniprep kit (Qiagen).

[0236] Example 12-2: Preparation of pCold-PS2K expression vector The linear DNA fragments of AzpK2 and AzpK2 Homolog1-2 purchased from Europhin Genomics in Example 12-1 were cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.) to obtain the respective inserted gene fragments.

[0237] Next, PCR was performed according to standard methods using primers of SEQ ID NOs: 164 and 165 for the pCold-ProS2 vector (Takara Bio Inc.), and primers of SEQ ID NOs: 166 and 167 for the pKW32 vector, to obtain the respective PCR-amplified fragments.

[0238] These PCR-amplified fragments were ligated using an In-Fusion® HD Cloning Kit (Takara Bio Inc.) according to standard methods. Subsequently, E. coli JM109 strain was transformed according to standard methods and cultured overnight at 30°C on LB (Km50) agar medium. Plasmid DNA was extracted from the resulting colonies by miniprep, and the drug resistance gene of the pCold-ProS2 vector was changed to kanamycin to obtain the pCold-PS2K vector.

[0239] The resulting pCold-PS2K vector was cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.), and then dephosphorylated in the same manner as in Example 12-1. Further, ligation was performed in the same manner as in Example 12-1 using each insert gene fragment of AzpK2 or AzpK2 Homolog1-2 to obtain the respective expression vectors, pCold-PS2K-AzpK2 and pCold-PS2K-1-2.

[0240] Example 12-3: Flask culture Using the expression vectors obtained in Examples 12-1 and 12-2, the pKW32 plasmid was introduced into E. coli JM109 strain, and the pCold-PS2K plasmid was introduced into E. coli BL21 star (DE3) (manufactured by Thermo Fisher Scientific) according to a standard method.

[0241] The obtained colonies were inoculated into 2 mL of LB (Km50) medium and cultured with shaking. 75 W / V% glycerol was added to the culture medium to a final concentration of 15 W / V% and mixed to prepare frozen bacterial stocks. The frozen bacterial stocks were stored at -80°C. Each frozen bacterial stock was inoculated into 2 mL of LB (Km50) medium and cultured overnight with shaking at 30°C and 180 rpm to prepare preculture solutions.

[0242] For E. coli JM109 / pKW32, 40 μL of the preculture was inoculated into 40 mL of LB (Km50) medium, and isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as "IPTG") was added to a final concentration of 0.2 mM. The mixture was cultured overnight at 30 ° C. and 230 rpm with shaking. For E. coli BL21 star (DE3) / pCold-PS2K, 1 mL of the preculture was inoculated into 40 mL of LB (Km50) medium and cultured at 30 ° C. and 230 rpm with shaking. After the OD630 nm reached 0.5, the mixture was left to stand at 15 ° C. for 30 minutes, and IPTG was added to a final concentration of 0.2 mM. The mixture was then cultured at 15 ° C. and 180 rpm with shaking for 24 hours.

[0243] Example 12-4: Reactivity evaluation using recombinant E. coli The culture medium obtained in Example 12-3 was centrifuged (6500 rpm, 10 minutes, 4°C) to collect cells. The wet cell weight of the collected cells was measured and suspended in 50 mM MES-NaOH buffer (pH 7.0) (hereinafter sometimes referred to as "MES buffer (pH 7.0)") to a concentration of 100 g / L, followed by ultrasonic disruption using an ultrasonic homogenizer, SONIFIER (registered trademark) 250D (manufactured by BRANSON). Using the resulting disrupted cell solution, reaction solutions were prepared in 14 mL Falcon (registered trademark) round tubes for each of reaction solution compositions 1 to 3 in Reaction Solution Composition Table 1.

[0244] In Table 1, the concentration of the bacterial cell lysate indicates the amount of bacterial cells collected by centrifugation. Hereinafter, expressions such as "··· g / L bacterial cell lysate" and "··· g / L bacterial cell suspension" have the same meaning.

[0245]

[0246] The prepared reaction solution was subjected to a reaction by shaking at 15° C. and 200 rpm for 19 hours using a three-stage constant temperature shaking incubator TAL-RS310 (manufactured by Thomas Scientific Instruments).

[0247] After 0.5 hours, 1 hour, 2 hours, and 4 hours had passed since the start of shaking (start of reaction), a portion of the reaction solution was sampled from each round tube, diluted 5-fold with purified water, and then ultrafiltered at 10,000×g for 10 minutes at room temperature using a filter unit (Amicon (registered trademark) Ultra-0.5 10 kDa (manufactured by Merck)). The obtained sample was analyzed by HPLC (analysis condition 7). Details of analysis condition 7 are as follows.

[0248]

[0249] The specific activity of the E. coli cell lysate expressing each hydroxylase was measured for the reaction solution prepared using reaction solution composition 1 in Reaction Solution Composition Table 1. The accumulated 5-hydroxy-L-lysine concentration in the sample after 0.5 hours of reaction for AzpK2, AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3, and after 4 hours of reaction for Pp_AzpK2, was measured and used as the 5-hydroxy-L-lysine-producing activity per gram of cells, i.e., specific activity. The specific activity is expressed in U / g-cell, with 1 unit (hereinafter referred to as "U") defined as the enzyme activity resulting in the accumulation of 1 μmol of 5-hydroxy-L-lysine per minute of hydroxylation reaction. The results of the specific activity measurements are shown in Table 9.

[0250]

[0251] As is clear from Table 9, AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3 exhibited higher specific activities than AzpK2 and Pp_AzpK2. Among them, AzpK2 Homolog 1-1 and AzpK2 Homolog 1-2 exhibited particularly high specific activities.

[0252] Regarding AzpK2 Homolog1-2, when the specific activities were compared using two types of cultured bacterial cells, the pKW32 vector (E. coli JM109 strain) and the pCold-PS2K vector (E. coli BL21 star (DE3) strain), the pKW32 vector (E. coli JM109 strain) showed a higher specific activity.

[0253] For AzpK2 Homolog 1-1, AzpK2 Homolog 1-2, and AzpK2 Homolog 1-3, the concentrations (accumulation amounts) of 5-hydroxy-L-lysine (5HL) in samples reacted for 1 hour in the reaction solutions prepared using reaction solution compositions 1 to 3 are compared. The results are shown in Table 10.

[0254] In Table 10, the relative values ​​are obtained by dividing the amount of 5-hydroxy-L-lysine accumulated by the amount of 5-hydroxy-L-lysine accumulated in the case of 20 mM L-Lys for each enzyme, and the terms "accumulated amount" and "accumulated concentration" have the same meaning.

[0255]

[0256] As is clear from Table 10, for both enzymes, the amount of 5-hydroxy-L-lysine accumulated was greatest when the L-Lys feed concentration was 20 mM, and the amount of 5-hydroxy-L-lysine accumulated decreased as the L-Lys feed concentration increased to 50 mM and 100 mM. This suggests that for both enzymes, the phenomenon of substrate inhibition occurred, in which reaction inhibition occurred and the amount of reaction product decreased as the feed concentration of L-Lys, the substrate, increased.

[0257] Here, the degree of influence of substrate inhibition on the reduction in reaction rate indicates that the higher the relative value when the substrate concentration is increased, the less the influence of substrate inhibition, and conversely, the lower the relative value when the substrate concentration is increased, the greater the influence of substrate inhibition.

[0258] The results in Table 10 show that AzpK2 Homolog 1-3 is the enzyme least affected by substrate inhibition, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 1-1 in that order.

[0259] Example 13 (Evaluation of activity of AzpK2 homologues using recombinant Escherichia coli - 2) Transformants of the E. coli BL21 star (DE3) strain were obtained according to standard methods using the pCold I vector (manufactured by Takara Bio Inc.) and the nine expression vectors pCold I-1-1 to 1-9 obtained in Example 3.

[0260] Each of the transformants of the pCold I vector and pCold I-1-1 to 1-9 was inoculated into 2 mL of Terrific Broth medium containing 100 μg / mL of sodium ampicillin (hereinafter referred to as "TB (Amp100) medium") and cultured with shaking at 30°C overnight (preculture).

[0261] 0.4 mL of each of the obtained preculture solutions was inoculated into 40 mL of another TB (Amp100) medium and cultured with shaking at 30° C. The culture was continued until the OD630 nm reached 0.5, and the mixture was left to stand at 15° C. for 30 minutes. IPTG was then added to a final concentration of 0.02 mM, and the mixture was further cultured with shaking at 15° C. and 200 rpm for 24 hours to obtain each culture solution.

[0262] A cell lysate was obtained from each of the culture solutions in the same manner as in Example 12-4. The resulting cell lysate was used to prepare a solution containing 20 mM L-Lys, 30 mM α-ketoglutaric acid, 2 mM sodium ascorbate, 0.5 mM FeSO ４ 0.4 mL of reaction solution was prepared with the following composition: 2.5 mM citric acid, 50 mM HEPES (pH 7.0), and 50 g / L of disrupted bacterial cell solution. Each of the resulting reaction solutions was shaken overnight at 15°C and 200 rpm using a small thermostatic shaking incubator BR-22FP (manufactured by Taitec Corporation).

[0263] Sampling was performed 1 hour, 2 hours, 4 hours, and 24 hours after the start of shaking (start of reaction), diluted 25-fold with purified water, and then ultrafiltered at 10,000×g for 10 minutes at room temperature using a filter unit (Amicon (registered trademark) Ultra-0.5 10 kDa (Merck)). The obtained samples were analyzed for the amount of 5-hydroxy-L-lysine accumulated by HPLC (analysis condition 8). Details of analysis condition 8 are as follows.

[0264]

[0265] The measurement results of the amount of 5-hydroxy-L-lysine accumulated in the cell lysates of recombinant E. coli expressing the negative control pCold I vector and each AzpK2 homolog are shown in Figure 6. In Figure 6, the vertical axis represents the 5-hydroxylysine concentration (mM).

[0266] As is clear from Figure 6, accumulation of 5-hydroxylysine was observed in all of AzpK2 Homologs 1-1 to 1-9 except for the negative control pCold I vector. Among AzpK2 Homologs 1-1 to 1-9, the amount of 5-hydroxy-L-lysine accumulated was highest in AzpK2 Homolog 1-3, followed by AzpK2 Homolog 1-6 and AzpK2 Homolog 1-2, and then AzpK2 Homolog 1-1, AzpK2 Homolog 1-5, and AzpK2 Homolog 1-4.

[0267] Example 14 (Evaluation of Reactivity of S-Selective Lysine Hydroxylase Using Genetically Recombinant Escherichia coli - 3) Using each cell lysate of recombinant Escherichia coli transformed with the pCold I vector, pCold I-1-2, pCold I-1-3, or pCold I-1-6 obtained in Example 13, the effect of substrate inhibition on the enzyme and the hydroxylation activity of 5-hydroxy-L-lysine were evaluated.

[0268] Example 14-1 Evaluation of the Effect of Substrate Inhibition on S-Selective Lysine Hydroxylase Reaction solutions were prepared using reaction compositions 4 to 7 in Reaction Solution Composition Table 2, and reactions were carried out in the same manner as in Example 13, and the accumulated concentration of 5-hydroxy-L-lysine was measured.

[0269]

[0270] For each of the reaction solutions reacted with reaction solution compositions 4 to 7, the accumulated amount and relative value of 5-hydroxy-L-lysine in the sample after 1 hour of reaction, which were measured and calculated in the same manner as in Example 13, are shown in Table 11.

[0271] In Table 11, the relative values ​​are calculated by dividing the amount of 5-hydroxy-L-lysine accumulated by the amount of 5-hydroxy-L-lysine accumulated when 20 mM L-Lys was added for each enzyme.

[0272]

[0273] As is clear from Table 11, the relative values ​​representing the influence of substrate inhibition were highest for AzpK2 Homolog 1-2, followed by AzpK2 Homolog 1-3 and AzpK2 Homolog 1-6, in that order. Therefore, it was found that the enzyme least susceptible to the influence of substrate inhibition was AzpK2 Homolog 1-2, followed by AzpK2 Homolog 1-3 and AzpK2 Homolog 1-6, in that order.

[0274] On the other hand, the amount of 5-hydroxy-L-lysine accumulated was highest in AzpK2 Homolog 1-3, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 1-6. Furthermore, the 5-hydroxy-L-lysine accumulation concentration, which indicates the productivity of 5-hydroxy-L-lysine, of AzpK2 Homolog 1-3 was higher than that of AzpK2 Homolog 1-2 and AzpK2 Homolog 1-6 under all conditions where the concentration of the substrate L-lysine was 20 to 100 mM. This indicates that although AzpK2 Homolog 1-3 is more susceptible to substrate inhibition than AzpK2 Homolog 1-2, its productivity of 5-hydroxy-L-lysine is superior to that of AzpK2 Homolog 1-2 and AzpK2 Homolog 1-6.

[0275] Example 14-2 Evaluation of 5-hydroxy-L-lysine hydroxylation activity of S-selective lysine hydroxylase L-lysine 5-hydroxylase has the activity of hydroxylating L-lysine, but also has the activity of further hydroxylating the 5-position of the product, 5-hydroxylysine, as shown in Example 10. Furthermore, if 5-oxolidine is produced here, the yield of the target product, 5-hydroxylysine, decreases. Therefore, when attempting to establish a production method for synthesizing 5-hydroxylysine, it is preferable that the hydroxylating activity of 5-hydroxylysine by the hydroxylase be low.

[0276] Therefore, the hydroxylation activity for L-lysine and the hydroxylation activity for 5-hydroxylysine were compared among AzpK2 Homolog 1-2, AzpK2 Homolog 1-3, and AzpK2 Homolog 1-6.

[0277] 20 mM racemic 5-hydroxylysine, 30 mM α-ketoglutaric acid, 2 mM ascorbic acid, 0.5 mM FeSO ４ 0.4 mL of a reaction solution was prepared containing 2.5 mM citric acid, 50 mM HEPES (pH 7.0), and 50 g / L of disrupted bacterial cell solution, and the reaction was carried out under the same conditions as in Example 13 except for the reaction time of 3 hours, and the 5-hydroxy-L-lysine concentration was measured. The measured 5-hydroxy-L-lysine concentration was divided by the reaction time to calculate the 5-hydroxy-L-lysine accumulation rate per unit time.

[0278] The remaining amount of 5-hydroxy-L-lysine after 3 hours of reaction with each enzyme was subtracted by the difference in the remaining amount of 5-hydroxy-L-lysine in the negative control strain introduced with pCold I vector to calculate the 5-hydroxy-L-lysine consumption rate per unit time.

[0279] The 5-hydroxy-L-lysine consumption rate was divided by the 5-hydroxy-L-lysine accumulation rate measured in Example 14-1 to calculate the 5-hydroxy-L-lysine / L-Lys hydroxylation activity ratio, which is shown in Table 12.

[0280]

[0281] The higher the 5-hydroxy-L-lysine / L-Lys hydroxylation activity ratio, the more likely it is that 5-oxolidine will be produced when 5-hydroxy-L-lysine is reacted as a substrate; therefore, a lower 5-hydroxy-L-lysine / L-Lys hydroxylation activity ratio can increase the 5-hydroxy-L-lysine yield. As is clear from Table 12, the 5-hydroxy-L-lysine / L-Lys hydroxylation activity ratio was lowest for AzpK2 Homolog 1-3, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 1-6 in that order. From this, it is considered that the 5-hydroxy-L-lysine yield was highest for AzpK2 Homolog 1-3, followed by AzpK2 Homolog 1-2 and AzpK2 Homolog 1-6 in that order.

[0282] Example 15 (Study of reaction conditions) Example 15-1 The pKW32-1-3 vector prepared in Example 12-1 was introduced into the E. coli JM109 strain according to a standard method to prepare the E. coli JM109-pKW32-1-3 strain. The resulting E. coli JM109-pKW32-1-3 strain was cultured according to a standard method, with enzyme expression induction using IPTG to a final concentration of 0.2 mM, to obtain cultured cells of the E. coli JM109-pKW32-1-3 strain.

[0283] Example 15-2: Investigation of cell disruption conditions The cultured cells of the E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in buffer solutions under the following conditions I to III to a concentration of 100 g / L, and then ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON).

[0284]

[0285] The resulting disrupted cell solution and the undisrupted cell suspension were used as reaction composition 5 of Example 14-1, and reacted at 15°C for 0.5 hours in the same manner as in Example 13. The accumulated concentration of 5-hydroxy-L-lysine in the resulting reaction solution was analyzed by HPLC (analysis condition 8). The analytical results are shown in Figure 7. In Figure 7, the vertical axis represents the 5-hydroxylysine concentration (mM).

[0286] 7, the highest concentration of 5-hydroxy-L-lysine accumulated was observed under condition II, where NaCl was added to the HEPES-pH buffer solution, followed by condition III, where NaCl and glycerol were added to the HEPES-pH buffer solution, and condition I, where only the HEPES-pH buffer solution was used. This suggests that the decrease in activity of L-lysine 5-hydroxylase upon cell disruption can be alleviated under condition II or III, where NaCl was added.

[0287] Example 15-3: Investigation of pH of reaction solution Using the cell lysate obtained under condition II in Example 15-2, reaction solutions were prepared with reaction compositions 7 to 13 in Table 3 of reaction solution composition, and reacted at 15°C for 0.5 hours in the same manner as in Example 13.

[0288]

[0289] The concentration of accumulated 5-hydroxy-L-lysine in the resulting reaction solution was analyzed by HPLC (analysis condition 8). The analysis results are shown in Figure 8. In Figure 8, the vertical axis represents the 5-hydroxylysine concentration (mM).

[0290] As is clear from FIG. 8, the same level of 5-hydroxy-L-lysine accumulation concentration was obtained in the reaction compositions with pH values ​​of 6.0 to 8.0, indicating that the reaction composition is hardly affected by pH in the range of pH 6.0 to 8.0.

[0291] Example 15-4: Substrate concentration study -1 Cultured cells of the E. coli JM109-pKW32-1-3 strain obtained in Example 15-1 were suspended in the buffer solution of Condition II in Example 15-2 to a concentration of 100 g / L or 200 g / L, and then ultrasonically disrupted using a SONIFIER (registered trademark) 250D (manufactured by BRANSON). Using each of the resulting disrupted cell solutions, reaction solutions were prepared according to Reaction Solution Composition Table 4, and each reaction was carried out at 15°C for 22.5 hours in the same manner as in Example 13.

[0292]

[0293] The reaction solution was sampled appropriately during the reaction, and the accumulated concentration of 5-hydroxy-L-lysine in the reaction solution was analyzed by HPLC (analysis condition 8). The analysis results are shown in FIG. 9. In FIG. 9, the vertical axis represents the 5-hydroxylysine concentration (mM) or the L-lysine concentration (mM). The accumulated concentration of 5-hydroxy-L-lysine was measured 22.5 hours after the start of the reaction, and was found to be 50 mM for the reaction with reaction composition 13, approximately 95 mM for the reaction with reaction composition 14, and approximately 180 mM for the reaction with reaction composition 15.

[0294] Example 15-5: Examination of substrate concentration -2 Using the disrupted bacterial cell solution obtained under condition II in Example 15-2, 0.35 L of reaction solution was prepared according to reaction compositions 16 and 17 in Table 5 of Reaction Solution Composition. Each reaction solution was reacted for 24 hours in a 1 L jar fermenter (manufactured by Biot) at 15°C, pH 7.0 (adjusted by adding 1 M HCl as appropriate), aeration rate of 0.35 L / min, dissolved oxygen concentration of 20%, and stirring speed of 400 to 1000 rpm (stirring speed adjusted so that the dissolved oxygen concentration would be 20% or higher).

[0295]

[0296] The reaction solution was sampled appropriately during the reaction, and the accumulated 5-hydroxy-L-lysine concentration in the reaction solution was analyzed by HPLC (analysis condition 7). The analysis results are shown in FIG. 10. In FIG. 10, the vertical axis represents the 5-hydroxylysine concentration (mM) or the L-lysine concentration (mM). Under the conditions of reaction composition 16, L-Lys was almost completely consumed, the accumulated 5-hydroxy-L-lysine concentration was 18.2 mM (2.95 g / L), and a highly purified 5-hydroxy-L-lysine solution containing approximately 1.0 g of 5-hydroxy-L-lysine was obtained. Under the conditions of reaction composition 17, 4.3 mM (0.62 g / L) of L-Lys remained, but the accumulated 5-hydroxy-L-lysine concentration was 40.6 mM (6.58 g / L), and a 5-hydroxy-L-lysine solution containing approximately 2.3 g of 5-hydroxy-L-lysine was obtained.

[0297] Example 16 Evaluation of Activity of L-lysine 5-Hydroxylase Homologs Belonging to Clades 2 to 4 Example 16-1 Preparation of pC1K Expression Vector Expressing L-lysine 5-Hydroxylase Homolog The pC1K vector, in which the drug resistance gene of the pCold I vector was changed to kanamycin, was obtained in the same manner as in Example 12-2, using primers represented by SEQ ID NOs: 164 and 165 for the pCold I vector (Takara Bio Inc.) and primers represented by SEQ ID NOs: 166 and 167 for the pKW32 vector.

[0298] The resulting pC1K vector was cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.), dephosphorylated in the same manner as in Example 12-1, and further cleaved with the following DNA fragments: SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15 Each of the linear DNA fragments 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, and 133 was cleaved with restriction enzymes NdeI and XhoI (both manufactured by Takara Bio Inc.). The resulting inserted gene fragments were ligated in the same manner as in Example 12-1 to obtain pC1K expression vectors expressing the L-lysine 5-hydroxylase enzymes shown in Table 13 below.

[0299]

[0300] Example 16-2: Evaluation of activity of L-lysine 5-hydroxylase belonging to Clades 2 to 4 The pC1K expression vector expressing the L-lysine 5-hydroxylase obtained in Example 16-1 was cultured in a flask under the same conditions as in Example 13, except that IPTG was added to a final concentration of 0.05 mM. The resulting cultured cells were suspended in 50 mM HEPES-NaOH buffer (pH 8.0) containing 200 mM NaCl to a concentration of 100 g / L, and then subjected to ultrasonic disruption using a SONIFIER® 250D (manufactured by BRANSON) to obtain a disrupted cell solution. The resulting disrupted cell solution was adjusted to reaction composition 4 of Example 14-1 and reacted at 15°C for 24 hours in the same manner as in Example 13.

[0301] The reaction mixture was sampled 24 hours after the start of the reaction, and the concentration of accumulated 5-hydroxy-L-lysine in the reaction mixture was analyzed by HPLC (analysis condition 8). Furthermore, pKW32-1-3 was used as a positive control, and the pC1K vector was used as a negative control, and the culture, cell disruption, and reaction were carried out in the same manner as for the pC1K expression vector expressing L-lysine 5-hydroxylase. The accumulated 5-hydroxy-L-lysine concentrations in each sample are shown in Table 14.

[0302] The symbols in Table 14 have the following meanings: -: 5-hydroxy-L-lysine was not detected *: 5-hydroxy-L-lysine accumulated at a concentration of 3 mM or less **: 5-hydroxy-L-lysine accumulated at a concentration of more than 3 mM and 10 mM or less ***: 5-hydroxy-L-lysine accumulated at a concentration of more than 10 mM

[0303]

[0304] As is clear from Table 14, the activity of L-lysine 5-hydroxylases belonging to Clades 2 to 4 was evaluated. As a result, AzpK2 Homolog-C3-4 and AzpK2 Homolog-C3-15 had the highest activity, followed by AzpK2 Homolog-C2-2, AzpK2 Homolog-C2-3, AzpK2 Homolog-C2-8, AzpK2 Homolog-C2-9, AzpK2 Homolog-C2-11, AzpK2 Homolog-C2-25, AzpK2 Homolog-C3-10, and AzpK2 Homolog-C3-12.

[0305] Example 17 (Comparison of Amino Acid Sequence Identity of L-lysine 5-hydroxylase) The L-lysine 5-hydroxylase for which S-selective L-lysine 5-hydroxylation activity was detected in Examples 10 and 16 was compared for amino acid sequence identity using ClustalW. The results are shown in FIG. 11 .

[0306] In FIG. 11, the areas with a gray background (red text and a pink background in the reference drawing) indicate areas with an identity value of 55% or more.

[0307] In FIG. 11, AzpK2 Homologs 1-1 to 1-9 belonging to Class 1 all share 55% identity with AzpK2.

[0308] From the results in FIG. 11, AzpK2 and AzpK2 Homologs 1-1 to 1-9 belonging to Class 1 all had identities of 55% or more when AzpK2 was used as the standard.

[0309] Furthermore, the L-lysine 5-hydroxylases in which R-selective L-lysine 5-hydroxylation activity was detected in Examples 10 and 16 were compared for amino acid sequence identity using ClustalW, and the results are shown in FIG. 12 .

[0310] In Figure 12, areas with a light gray background (light blue in the reference drawing) indicate areas with an identity value of 58% or more, and areas with a dark gray background (yellow in the reference drawing) indicate areas with an identity value of 50% or more.

[0311] 12, it was found that the amino acid sequences having L-lysine 5-hydroxylation activity belonging to Clade 2 and Clade 3 share 50% or more identity with AzpK2 Homolog C2-22. Furthermore, it was found that the amino acid sequences having L-lysine 5-hydroxylation activity in Clade 4 share 58% or more identity with each other.

[0312] Example 18 (Crystal Structure Analysis of AzpK2) Example 18-1: Purification of AzpK2 Protein An aqueous solution of recombinant AzpK2 protein obtained by the same method as in Example 4 was purified by anion exchange chromatography using AKTA pure (Cytiva). Elution was performed using a NaCl concentration gradient (solution A (20 mM HEPES, 10% W / V glycerol, pH 8.0) and solution B (20 mM HEPES, 10% W / V glycerol, 1 M NaCl, pH 8.0)), and a RESOURCE® Q 1 mL column (Cytiva) was used. The solvent of the solution containing the eluted protein was replaced with solution A by ultrafiltration (Amicon Ultra-15 10 kDa, Merck), and the protein concentration was adjusted to 10 mg / mL to obtain a purified solution of AzpK2 protein.

[0313] Example 18-2 Preparation of AzpK2 Protein Crystals Initial screening was performed using crystallization screening kits Crystal Screen (Hampton Research), Index (Hampton Research), Wizard I (Molecular Dimensions), and Wizard II (Molecular Dimensions).

[0314] In the screening, 1 μL of crystallization buffer and protein solution were mixed and allowed to stand at 20°C for crystallization using the sitting drop method. As a result, it was found that good crystals could be obtained under several conditions. Therefore, for the conditions under which good crystals were obtained, 2 μL of crystallization buffer and protein solution were mixed and crystallized using the hanging drop method. The pH and amount of precipitant were examined. As a result, the best AzpK2 single crystals were obtained under the conditions in which the crystallization buffer contained 24 W / V% PEG3350, 100 mM Bis-Tris pH 6.5, and 200 mM NaCl.

[0315] Next, we attempted to obtain a co-crystal with 5-hydroxy-L-lysine and α-ketoglutaric acid in the same manner as above. 5-hydroxy-L-lysine and α-ketoglutaric acid were added to the storage buffer of AzpK2 to a concentration of 1 mM, and the mixture was left to stand on ice for 30 minutes, after which crystallization was carried out by the hanging drop method in the same manner as above. As a result, co-crystals were obtained using a storage buffer with a composition of 22 W / V % PEG3350, 100 mM Bis-Tris pH 6.5, and 200 mM NaCl.

[0316] Example 18-3: X-ray structural analysis of AzpK2 protein crystals The AzpK2 single crystals obtained in Example 18-2 were scooped up with a litho loop and frozen in liquid nitrogen. Then, using the beamline PF BL1A at the High Energy Accelerator Research Organization, the frozen crystals were irradiated with X-rays under a nitrogen gas flow at 100 K to obtain diffraction patterns. As a result, a diffraction pattern with a maximum resolution of 1.60 angstroms was obtained.

[0317] The data were processed using XDS software (Max Planck Institute for Medical Research) and further scaled using AIMLESS software. Phase determination by molecular replacement was performed using Phenix phaser, and an initial model was constructed using AutoBuild.

[0318] Based on the initial model constructed, a structural model was created by repeatedly adjusting the structure manually using COOT and refining using phenix.refine. Due to the sparse electron density, some models (Figure 13, left, (1) Gly60 to Met78, (2) Arg132 to His136, (3) Asp230 and beyond) could not be constructed, and the region thought to be the active center of the enzyme was exposed.

[0319] Next, a similar experiment was performed using a cocrystal of 5-hydroxy-L-lysine and α-ketoglutaric acid, resulting in a diffraction pattern with a maximum resolution of 2.24 Å. The obtained data was processed in the same way, and a model was constructed. Although the electron density corresponding to 5-hydroxy-L-lysine and α-ketoglutaric acid could not be clearly observed, a model could be constructed for regions that were not observed in the structure without substrate binding (Figure 13, right). However, the structure beyond Glu236, a part of the C-terminus, was not observed, and the substrate binding mode could not be fully elucidated.

[0320] The left figure in Figure 13 is a structural model obtained from a single crystal of AzpK2, and the right figure is a structural model obtained from a crystal of AzpK2 with 5-hydroxy-L-lysine and α-ketoglutarate. The ribbon represents the AzpK2 protein, and the gray spheres (orange in the reference drawing) represent iron atoms.

[0321] Example 19 (Identity Comparison between AzpK2 and BesD) The ClustalW method was used to compare amino acid identities between AzpK2, which belongs to Clade 1, and BesD, which belongs to Clade 5. The results of the identity comparison are shown in Figure 14.

[0322] BesD has a sequence relatively similar to that of AzpK2 and is an αKG-dependent dioxygenase that uses the same lysine as a substrate, but its function differs from that of AzpK2 in that it halogenates lysine. Non-Patent Document 8 reports the positions of amino acid residues important for the activity of BesD.

[0323] As is clear from FIG. 14, in AzpK2, as in BesD, the amino acid residues that interact with α-ketoglutaric acid, iron atoms, the α-amino group of lysine as a substrate, and carboxylic acid are conserved.

[0324] In Figure 14, ○ indicates a residue that interacts with the carboxylic acid of L-lysine, ● indicates a residue that interacts with the iron atom, ◎ indicates a residue that interacts with the water molecule coordinated around the α-amino group of L-lysine, and ▽ indicates a residue that interacts with αKG.

[0325] Example 20 (Identification of important motifs for enzyme activity by predicting the three-dimensional structure of AzpK2) The three-dimensional structure of AzpK2 was predicted using AlphaFold2, a protein structure prediction software, and it was possible to predict the structure of even parts for which an X-ray structure analysis model could not be constructed, as shown in Figure 15. Furthermore, the predicted structure of AzpK2 was compared with the three-dimensional structure of BesD, the three-dimensional structure of which has been reported.

[0326] As a result, the parts that could not be modeled by X-ray structural analysis all corresponded to amino acid residues that were close to the substrates L-lysine, αKG, or iron atoms and were predicted to be important for the reaction.

[0327] In Figure 15, the upper left figure shows the results of model construction (near the active center) by X-ray structural analysis of an AzpK2 single crystal, the upper right figure shows the results of model construction (near the active center) by X-ray structural analysis of a co-crystal of AzpK2, αKG, and 5-hydroxylysine, the lower left figure shows the predicted structure of BesD in AlphaFold2, and the lower right figure shows the predicted structure of AzpK2 in AlphaFold2 (BesD-derived lysine and αKG are superimposed).

[0328] In each figure in Figure 15, the gray sphere near the center (orange sphere in the reference figure) represents the region of high electron density. In αKG-dependent dioxygenases, the iron atom (Fe ２＋ ) is known to be the corresponding ion. The light gray sphere near the center (the green sphere in the reference drawing) represents the iron atom ion in BesD.

[0329] Specifically, in Figure 15, region (2) of AzpK2 corresponds to the region close to the carbonyl group and amino group of L-lysine and αKG in BesD. In particular, the His136-Asp141 region is suggested to be close to the amino group of lysine, carboxylic acid, or iron atom, indicating that it is an important region that contributes to substrate selectivity and activity.

[0330] Next, the region (3) of AzpK2, particularly the region from Thr237 to Phe242, corresponds to the region close to the alkyl chain at positions 3 to 5 of L-lysine, which is modified by the enzymatic reaction in BesD, as shown in Figure 15. This region was shown to be important in determining the modification site of lysine and the optical selectivity of the hydroxyl group.

[0331] Furthermore, when amino acid residues within the enzyme interact with factors other than the enzyme (e.g., substrate, αKG, iron atom, etc.), it is known that these interactions are influenced by the properties of the adjacent amino acid residues (e.g., molecular size, electrical properties, etc.). Three-dimensional structure prediction suggested that Asp142 in region (2) and His235 and Asn236 in region (3) may be involved in the interaction.

[0332] From the above, it was revealed that in the L-lysine 5-hydroxylation activity of AzpK2, the amino acid residues important for the enzymatic activity are His136 to Asp142, and the amino acid residues important for determining the optical selectivity of the L-lysine 5-hydroxylation activity are His235 to Phe242.

[0333] Example 21 (Measurement of activity of amino acid substitution mutants of AzpK2) Using pCold I-AzpK2 prepared in Example 1 as a template, the primer set for introducing amino acid substitution mutations shown in Table 15 was used, and PrimeStar HS DNA polymerase (manufactured by Takara Bio Inc.) was used in accordance with a standard method to obtain pCold I-AzpK2 vectors encoding the corresponding amino acid substitution mutants.

[0334]

[0335] For the resulting amino acid-substituted AzpK2, an aqueous protein solution of each AzpK2 amino acid-substituted AzpK2 was prepared in the same manner as in Example 4. Using the resulting protein solution, an enzyme assay was performed in the same manner as in Example 5, and the Fmoc-conjugated sample was analyzed by LC-MS (analysis condition 2). The analysis results are shown in Figure 16. In Figure 16, WT represents wild-type AzpK2.

[0336] As shown in Figure 16, 5-hydroxylysine was not detected in the amino acid substituted AzpK2 mutants (R76A, E122A, H136A, D141G, and F242W), indicating that the activity was lost, suggesting that these amino acid residues are important for the activity.

[0337] Example 22 (Identification of important motifs for enzymatic activity in AzpK2 and homologs) The important amino acid residues related to the enzymatic activity of AzpK2, which were identified in Example 20, were compared with the amino acid sequences of Pp_AzpK2, which belongs to Clades 1 to 4 and Clade 10, which are L-lysine 5-hydroxylase enzymes, and Clade 5, which is close to Clade 4 but does not have L-lysine 5-hydroxylation activity.

[0338] First, the identity of the amino acid sequences belonging to Clades 1 to 5 was compared within each Clade using ClustalW. Among the results of this identity comparison, the amino acid sequences of His136 to Asp142 and His235 to Phe242, which are important regions of AzpK2 identified in Example 20, are shown in Figures 17 and 18 as logo plots of the occurrence frequencies of amino acids in each of the amino acid sequences belonging to Clades 1 to 5.

[0339] Furthermore, for Pp_AzpK2, the amino acid sequence of Pp_AzpK2 was compared with the amino acid sequences belonging to Clades 1 to 5 without comparing identity within the Clade.

[0340] As a result of comparing the amino acids at positions 136 to 142, as shown in Figure 17, the amino acids at positions 136 to 142 belonging to Clades 1 to 4 all conserved the same amino acids, and the sequence of amino acids at positions 136 to 142 was "HGWHWDD".

[0341] Furthermore, the amino acids at positions 136 to 142 of Pp_AzpK2 were "HGWHLDD", while the amino acids at positions 136 to 142 of Clade 5 were "HGWHWGD".

[0342] Here, Non-Patent Document 8 reports that the combination of amino acids at positions 139 and 141 is characterized as "HXD" in hydroxylases and "HXG" in halogenases. Furthermore, Non-Patent Document 8 also reports that the "H" located at the third amino acid N-terminally of "HXD" or "HXG" is important for enzyme activity. Furthermore, Example 21 above also reveals that introducing an amino acid mutation into His136 of AzpK2 results in a loss of enzyme activity.

[0343] This suggests that the "HGWHWDD" or "HGWHLDD" motif, which is a group of amino acid residues from His136 to Asp142, is important for the expression of the hydroxylation activity of L-lysine 5-hydroxylase.

[0344] Next, the results of comparing the amino acids at positions 235 to 242 are shown in Figure 18. Furthermore, from the results of Figure 18, amino acids that are not conserved within the same Clade are indicated by "X," and the results of comparing the amino acids at positions 235 to 242 of Clades 1 to 5 and Pp_AzpK2 are shown in Table 16.

[0345]

[0346] From the results of Example 20, it can be said that the region of amino acids 235 to 242, which plays an important role in determining the optical selectivity of the L-lysine 5-hydroxylation activity, is itself a motif, a group of amino acid residues.

[0347] Therefore, for Clades 1 to 4, the amino acids of the motif were identified based on the results of identity comparison.

[0348] First, amino acids 235 and 236 are completely conserved among Clades 1 to 4. Next, for amino acid 237, threonine (T) is conserved except for one, and one of the amino acids belonging to Clade 3 is glycine (G), but its activity is relatively low among Clade 3, and threonine (T) can be identified as the conserved amino acid.

[0349] Amino acid 238 was conserved as methionine (M) in Clades 1 to 3 and asparagine (N) in Clade 4. Amino acid 239 was conserved as aspartic acid (D), glutamic acid (E), or asparagine (N) in Clades 1 to 4, showing a certain degree of conservation: glutamic acid (E) in Clade 1, glutamic acid (E) or aspartic acid (D) in Clades 2 to 3, and aspartic acid (D) or asparagine (N) in Clade 4.

[0350] The amino acid at position 240 is not conserved among Clades 1 to 4. The amino acids at positions 241 and 242 are conserved as follows: leucine (L) + phenylalanine (F) in Clade 1, leucine (L) + tryptophan (W) in Clades 2 and 3, and leucine (L) + phenylalanine (F) in Clade 4.

[0351] From the above, the "HETMEXLF" motif was identified as a conserved motif in Clade 1, which has S-selective L-lysine 5-hydroxylation activity, while the "HETMDXLW" or "HETMEXLW" motif was identified in Clade 2 and Clade 3, which have R-selective L-lysine 5-hydroxylation activity, and the "HETNDXLF" or "HETNNXLF" motif was identified in Clade 4, which also has R-selective L-lysine 5-hydroxylation activity. Furthermore, Pp_AzpK2 has an amino acid sequence with characteristics different from the motifs of Clades 1 to 4, and Clade 5, which is close but does not have L-lysine 5-hydroxylation activity, has an amino acid sequence different from both Clades 1 to 4 and Pp_AzpK2.

[0352] In Example 21, the F242W mutant of AzpK2 lost the activity of hydroxylating L-lysine at position 5, and the optical selectivity of the hydroxyl group of L-lysine could not be changed even when only the 242nd amino acid, which differs between Clade 1 and Clade 2, was replaced.

[0353] This suggests that not only one amino acid constituting amino acids 236-242 but also the motif of amino acids 236-242 is important for the functional expression of L-lysine 5-hydroxylase.

[0354] Example 23 (Search for a gene having L-lysine 5-hydroxylase activity) A search for a gene having L-lysine 5-hydroxylase activity was carried out based on the information in Non-Patent Document 7. Specifically, based on the genetic information of the Streptacidiphilus griseoplanus strain and the Kitasatospora azatica strain, which have the arazopeptin biosynthetic gene cluster described in Non-Patent Document 7, a search was conducted for a microorganism having an azp gene cluster.

[0355] As a result, as shown in Figure 1, we found that the homologous clusters of the azp gene clusters of Actinosynnema pretiosum strain and Actinosynnema mirum strain DSM 43827 strain do not contain the azpK gene, but instead contain a 2-oxoglutarate-dependent hydroxylase gene of unknown function (indicated by * in Figure 1).

[0356] This 2-oxoglutarate-dependent hydroxylase was named AzpK2, and the DNA encoding the AzpK2 protein was named azpK2 gene.

[0357] Furthermore, we similarly searched for the biosynthetic genes for 6-diazo-5-oxo-L-norleucine (azpJ, azpK, azpL) present in the azp gene cluster, and found that in the Pseudomonas psychotolerans NS383 strain, there is an azpK2 gene homolog with unknown function between the azpJ and azpL genes (indicated by a star in Figure 1).

[0358] This was named the Pp_azpK2 gene, and the protein encoded by the Pp_azpK2 gene was named Pp_AzpK2.

[0359] Figure 1 is a diagram comparing azp gene clusters. The meanings of the symbols in Figure 1 are as explained above.

[0360] As shown in the above examples, the AzpK2 protein had the activity of S-selectively hydroxylating the 5-position of L-lysine. Furthermore, the Pp_AzpK2 protein had the activity of R-selectively hydroxylating the 5-position of L-lysine. Furthermore, when 5-hydroxy-L-lysine was synthesized from L-lysine using transformants with DNA encoding the AzpK2 protein (azpK2 gene) and DNA encoding the Pp_AzpK2 protein (Pp_azpK2 gene), 5-hydroxy-L-lysine was successfully synthesized at a high accumulated concentration.

[0361] Example 24 (Search for AzpK2 and Pp_AzpK2 Homologs) Homology searches were performed for the azpK2 gene and the Pp_azpK2 gene using databases such as the DNA Databank of JAPAN (DDBJ), and gene sequence information for 1,000 homologous genes of each of the azpK2 gene and the Pp_azpK2 gene was obtained.

[0362] Next, overlapping homologous genes of the azpK2 gene and homologous genes of the Pp_azpK2 gene were eliminated to obtain gene sequence information of 1,115 homologous genes including the azpK2 gene and the Pp_azpK2 gene.

[0363] The amino acid sequences encoded by these homologous genes were classified based on a molecular phylogenetic tree created using the Neighbor-joining method, and were classified into 10 Clades. Each classified Clade was named Clades 1 to 10. Representative proteins encoded by the 1,115 homologous genes (including the azpK2 and Pp_azpK2 genes) classified into the 10 Clades are shown in Figure 2.

[0364] As shown in the above examples, proteins belonging to Clades 1 to 4 have L-lysine 5-hydroxylase activity. Of these, proteins belonging to Clade 1 have the activity of S-selectively hydroxylating the 5-position of L-lysine, and proteins belonging to Clades 2 to 4 have the activity of R-selectively hydroxylating the 5-position of L-lysine.

[0365] The L-lysine 5-hydroxylase and enzyme composition of the present invention can be used for producing 5-hydroxy-L-lysine, which is useful as a pharmaceutical intermediate, etc. Furthermore, according to the method for producing 5-hydroxy-L-lysine of the present invention, 5-hydroxy-L-lysine can be produced from L-lysine in high yield and at low cost. Furthermore, by using different L-lysine 5-hydroxylase enzymes, the desired (2S,5S)-5-hydroxy-L-lysine or (2S,5R)-5-hydroxy-L-lysine can be produced.

Claims

1. L-Lysine 5-hydroxylase comprising a polypeptide shown in any of the following (A), (B), (C), (D), (E) or (F): (A) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylase activity; (C) a polypeptide having an amino acid sequence having 55% or more sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having L-lysine 5-hydroxylase activity; (D) a polypeptide having an amino acid sequence having 50% or more sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having L-lysine 5-hydroxylase activity; (E) a polypeptide having an amino acid sequence having 58% or more sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-hydroxylase activity; (F) the motif of a continuous amino acid sequence "HGWHWDD";and a motif of any continuous amino acid sequence selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF", and "HETNNXLF"; and a polypeptide having L-lysine 5-position hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).; 2. DNA encoding L-lysine 5-hydroxylase according to claim 1.

3. The DNA according to claim 2, wherein the DNA is any one of the following (G), (H), (I), (J), (K), or (L): (G) DNA containing the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133; (H) DNA having a base sequence in which one or more bases are substituted, deleted, and / or added in the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (I) DNA encoding a polypeptide having 55% or more sequence identity with the polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (J) DNA encoding a polypeptide having 50% or more sequence identity with the polypeptide encoded by the base sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (K) DNA encoding a polypeptide having 58% or more sequence identity with the polypeptide encoded by the base sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (L) "HGWHWDD", which is a motif of a continuous amino acid sequence.and a motif of any contiguous amino acid sequence selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF", and "HETNNXLF"; and a DNA encoding a polypeptide having L-lysine 5-hydroxylase activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).; 4. A method for producing 5-hydroxy-L-lysine, characterized by contacting L-lysine with a polypeptide shown in any of the following (A), (B), (C), (D), (E) or (F), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell to produce 5-hydroxy-L-lysine: (A) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-position hydroxylation activity; (C) a polypeptide having an amino acid sequence having 55% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having L-lysine 5-position hydroxylase activity; (D) a polypeptide having an amino acid sequence having 50% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having L-lysine 5-position hydroxylation activity;(E) a polypeptide having an amino acid sequence having 58% or more identity with the amino acid sequence shown in SEQ ID NOs: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134 and having L-lysine 5-hydroxylation activity; (F) the motif of the continuous amino acid sequence "HGWHWDD"; and any continuous amino acid sequence motif selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLW" and having L-lysine 5-hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).; 5. The method for producing 5-hydroxy-L-lysine according to claim 4, wherein the cell is a cell transformed with the DNA encoding L-lysine 5-hydroxylase.

6. The method for producing 5-hydroxy-L-lysine according to claim 5, wherein the DNA is any one of the following (G), (H), (I), (J), (K) or (L): (G) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133; (H) DNA having a nucleotide sequence in which one or more bases are substituted, deleted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 57, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (I) DNA having 55% or more sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19 or 21, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (J) DNA having 50% or more sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (K) DNA having 58% or more sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having L-lysine 5-position hydroxylation activity;(L) "HGWHWDD", which is a motif of a continuous amino acid sequence; and any motif of a continuous amino acid sequence selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLW", and "HETNNXLW"; and DNA encoding a polypeptide having L-lysine 5-hydroxylase activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).; 7. An enzyme agent composition comprising a polypeptide shown in any of the following (A), (B), (C), (D), (E) or (F), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, and having L-lysine 5-position hydroxylation activity: (A) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-position hydroxylation activity; (C) a polypeptide having an amino acid sequence having 55% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having L-lysine 5-position hydroxylase activity; (D) a polypeptide having an amino acid sequence having 50% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having L-lysine 5-position hydroxylation activity; (E) a polypeptide having an amino acid sequence having 58% or more identity with the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having L-lysine 5-position hydroxylation activity;(F) a motif of a continuous amino acid sequence, "HGWHWDD"; and any motif of a continuous amino acid sequence selected from the group consisting of "HETMEXLF", "HETMDXLW", "HETMEXLW", "HETNDXLF", and "HETNNXLF"; and having L-lysine 5-position hydroxylation activity, wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

8. An S-selective L-lysine 5-hydroxylase comprising a polypeptide shown in the following (A-1), (B-1), (C-1) or (F-1): (A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22; (B-1) an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylase activity; (C-1) having an amino acid sequence having 55% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylase activity; (F-1) a polypeptide having the motifs of "HGWHWDD" and "HETMEXLF" which are consecutive amino acid sequences, and having S-selective L-lysine 5-hydroxylase activity (wherein X represents any one of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

9. DNA encoding the S-selective L-lysine 5-hydroxylase according to claim 8.

10. The DNA according to claim 9, wherein the DNA is any one of the following (G-1), (H-1), (I-1), or (L-1): (G-1) DNA containing the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21; (H-1) DNA having a base sequence in which one or more bases are substituted, deleted, and / or added in the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (I-1) DNA having a sequence identity of 55% or more with the polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having S-selective L-lysine 5-position hydroxylation activity; (L-1) DNA encoding a polypeptide having the motifs of "HGWHWDD" and "HETMEXLF" which are motifs of a continuous amino acid sequence, and having S-selective L-lysine 5-position hydroxylation activity (wherein X represents any one of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

11. A method for producing (2S,5S)-5-hydroxy-L-lysine, characterized in that L-lysine is brought into contact with a polypeptide shown in any of the following (A-1), (B-1), (C-1) or (F-1), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell to produce (2S,5S)-5-hydroxy-L-lysine: (A-1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22; (B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-position hydroxylation activity; (C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-position hydroxylase activity; (F-1) a polypeptide having the motifs of "HGWHWDD" and "HETMEXLF" which are consecutive amino acid sequences, and retaining S-selective L-lysine 5-position hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

12. The method for producing (2S,5S)-5-hydroxy-L-lysine according to claim 11, wherein the cell is a cell transformed with DNA encoding the L-lysine 5-position hydroxylase.

13. The method for producing (2S,5S)-5-hydroxy-L-lysine according to claim 12, wherein the DNA is any one of the following (G-1), (H-1), (I-1), or (L-1): (G-1) DNA containing the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21; (H-1) DNA having a base sequence in which one or more bases are substituted, deleted, and / or added in the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having L-lysine 5-position hydroxylation activity; (I-1) DNA having 55% or more sequence identity with the polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and encoding a polypeptide having S-selective L-lysine 5-position hydroxylation activity; (L-1) DNA encoding a polypeptide having the motifs of "HGWHWDD" and "HETMEXLF" which are consecutive amino acid sequences, and having S-selective L-lysine 5-position hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).

14. An enzyme composition comprising a polypeptide shown in the following (A-1), (B-1), (C-1) or (F-1), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, and having S-selective L-lysine 5-hydroxylation activity: (A-1) a polypeptide having an amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22; (B-1) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylation activity; (C-1) a polypeptide having an amino acid sequence having 55% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, 12, 14, 16, 18, 20 or 22, and having S-selective L-lysine 5-hydroxylase activity; (F-1) a polypeptide having the motifs of "HGWHWDD" and "HETMEXLF" which are consecutive amino acid sequences, and retaining S-selective L-lysine 5-hydroxylation activity (wherein X represents any one of the amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).

15. An R-selective L-lysine 5-hydroxylase comprising a polypeptide shown in any of the following (A-2), (B-2), (D-2), (E-2) or (F-2): (A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B-2) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylase activity; (D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having R-selective L-lysine 5-hydroxylase activity; (E-2) a polypeptide having an amino acid sequence having 58% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylase activity; (F-2) the motif of the continuous amino acid sequence "HGWHWDD"; and any motif of the continuous amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLW" and "HETNNXLW".A polypeptide having and retaining the R-selective L-lysine 5-hydroxylation activity, wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V); 16. DNA encoding the R-selective L-lysine 5-hydroxylase according to claim 15.

17. The DNA according to claim 16, wherein the DNA is any one of the following (G-2), (H-2), (J-2), (K-2), or (L-2): (G-2) DNA comprising the nucleotide sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133; (H-2) DNA having a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added in the nucleotide sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (J-2) DNA encoding a polypeptide having at least 50% sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (K-2) DNA encoding a polypeptide having at least 58% sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (L-2) a motif of a continuous amino acid sequence "HGWHWDD"; and any motif of a continuous amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLW", and "HETNNXLW".DNA encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity and having (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V); 18. A method for producing (2S,5R)-5-hydroxy-L-lysine, which comprises contacting L-lysine with a polypeptide shown in any of the following (A-2), (B-2), (D-2), (E-2) or (F-2), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell to produce (2S,5R)-5-hydroxy-L-lysine: (A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B-2) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective 5-position hydroxylation activity of L-lysine; (D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having R-selective 5-position hydroxylation activity of L-lysine; (E-2) a polypeptide having an amino acid sequence having 58% or more sequence identity with the full-length amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective 5-position hydroxylation activity of L-lysine; (F-2) the motif of a continuous amino acid sequence "HGWHWDD";and a motif of any continuous amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLF", and "HETNNXLF"; and has polypeptide that retains R-selective L-lysine 5-hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V).; 19. The method for producing (2S,5R)-5-hydroxy-L-lysine according to claim 18, wherein the cell is a cell transformed with the DNA encoding the L-lysine 5-hydroxylase.

20. The method for producing (2S,5R)-5-hydroxy-L-lysine according to claim 19, wherein the DNA is any one of the following (G-2), (H-2), (J-2), (K-2) or (L-2): (G-2) DNA comprising the nucleotide sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133; (H-2) DNA having a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added in the nucleotide sequence shown in SEQ ID NO: 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (J-2) DNA encoding a polypeptide having at least 50% sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 55, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (K-2) DNA encoding a polypeptide having at least 58% sequence identity with the polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 111, 113, 115, 119, 121, 123, 125, 127, 129, 131 or 133, and encoding a polypeptide having R-selective L-lysine 5-hydroxylation activity; (L-2) the motif of the continuous amino acid sequence "HGWHWDD"; and any motif of the continuous amino acid sequence selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLW" and "HETNNXLW".DNA encoding a polypeptide having R-selective L-lysine 5-hydroxylase activity, wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V); 21. An enzyme agent composition comprising a polypeptide shown in any of the following (A-2), (B-2), (D-2), (E-2) or (F-2), a cell containing the same, a preparation of the cell, or a culture solution obtained by culturing the cell, and having R-selective L-lysine 5-hydroxylation activity: (A-2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134; (B-2) a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity; (D-2) a polypeptide having an amino acid sequence having 50% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and having R-selective L-lysine 5-hydroxylation activity; (E-2) a polypeptide having an amino acid sequence having 58% or more sequence identity with the full length of the amino acid sequence shown in SEQ ID NO: 112, 114, 116, 120, 122, 124, 126, 128, 130, 132 or 134, and having R-selective L-lysine 5-hydroxylation activity; (F-2) a continuous amino acid sequence motif of "HGWHWDD"; and any continuous amino acid sequence motif selected from the group consisting of "HETMDXLW", "HETMEXLW", "HETNDXLW" and "HETNNXLW";A polypeptide having and retaining the R-selective L-lysine 5-hydroxylation activity (wherein X represents any amino acid of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V).;

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