L-lysine hydroxylase and production method for hydroxy-L-lysine and hydroxy-L-pipecolic acid using same

1. A method for producing hydroxy-L-lysine, said method comprising reacting free L-lysine with 2-oxoglutarate-dependent L-lysine hydroxylase, an isolated cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase, a processed product of said cell, wherein the processed product comprises the 2-oxoglutarate-dependent L-lysine hydroxylase, and/or a culture broth comprising the 2-oxoglutarate-dependent L-lysine hydroxylase obtained by culturing said cell, in an aqueous medium, or a mixture of the aqueous medium and an organic solvent that contains the free L-lysine, to convert the free L-lysine to hydroxy-L-lysine represented by the following General Formula (I):

and

collecting the hydroxy-L-lysine,

wherein each of R 1 , R 2 and R 3 represents a hydrogen atom or hydroxyl group, with the proviso that at least one of R 1 , R 2 and R 3 represents a hydroxyl group; and

wherein said 2-oxoglutarate-dependent L-lysine hydroxylase comprises a polypeptide selected from the group consisting of:

(A) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, or 12;

(B) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, or 12 except that at least one and not more than 10 amino acids are deleted, substituted, and/or added, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity; and

(C) a polypeptide comprising an amino acid sequence with a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity.

2. The method for producing hydroxy-L-lysine according to claim 1 , wherein said 2-oxoglutarate-dependent L-lysine hydroxylase comprises a polypeptide selected from the group consisting of:

(A) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 6, 8, or 10;

(B) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 6, 8, or 10 except that at least one and not more than 10 amino acids are deleted, substituted, and/or added, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity; and

(C) a polypeptide comprising an amino acid sequence with a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:2, 6, 8, or 10, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity.

3. The method for producing, hydroxy-L-lysine according to claim 1 , wherein

the free L-lysine is reacted with the isolated cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase, the processed product of said cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase, and/or the culture broth comprising the 2-oxoglutarate-dependent L-lysine hydroxylase obtained by culturing said cell; and

said cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase is a cell transformed with a DNA encoding the 2-oxoglutarate-dependent L-lysine hydroxylase.

4. The method for producing hydroxy-L-lysine according, to claim 3 , wherein said DNA encoding the 2-oxoglutarate-dependent L-lysine hydroxylase comprises a DNA selected from the group consisting of:

(A) a DNA comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, or 11;

(B) a DNA comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, or 11 except that at least one and not more than 30 nucleotides are substituted, deleted, and/or added, wherein the DNA encodes a polypeptide having 2-oxoglutarate-dependent L-lysine hydroxylase activity; and

(C) a DNA comprising a nucleotide sequence which hybridizes with the full complementary strand of the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, or 11 under stringent conditions comprising washing with 0.1×SSC and 0.1% SDS at 60° C., wherein the DNA encodes a polypeptide having 2-oxoglutarate-dependent hydroxylase activity.

5. The method for producing hydroxy-L-lysine according to claim 1 , wherein the free L-lysine is reacted with said 2-oxoglutarate-dependent L-lysine hydroxylase, the isolated cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase, the processed product of said cell comprising the 2-oxoglutarate-dependent L-lysine hydroxylase, and/or the culture broth comprising the 2-oxoglutarate-dependent L-lysine hydroxylase obtained by culturing said cell in the presence of 2-oxoglutaric acid and ferrous ion.

6. A method for producing hydroxy-L-pipecolic acid, said method comprising:

producing, hydroxy-L-lysine by the method of claim 1 ;

reacting the collected hydroxy-L-lysine with: (i) at least one enzyme selected from the group consisting of L-amino acid oxidase, L-amino acid dehydrogenase, and L-amino acid transferase, or (ii) amino acid racemase and at least one enzyme selected from the group consisting of D-amino acid oxidase, D-amino acid dehydrogenase, and D-amino acid transferase, to produce a compound represented by the following General Formula (II):

wherein R 1 , R 2 , and R 3 have the same meanings as in the General Formula (I) of claim 1 ; and

reacting the compound represented by the General Formula (II) with N-methyl-L-amino acid dehydrogenase to produce hydroxy-L-pipecolic acid represented by the following General Formula (III):

wherein R 1 , R 2 , and R 3 have the same meanings as in the General Formula (I) of claim 1 .

7. A method for producing hydroxy-L-pipecolic acid, said method comprising:

producing hydroxy-L-lysine by the method of claim 1 ;

reacting the collected hydroxy-L-lysine with at least one enzyme selected from the group consisting of L-lysine 6-oxidase, 6-dehydrogenase, and L-lysine 6-transferase, to produce a compound represented by the following General Formula (IV):

wherein R 1 , R 2 , and R 3 have the same meanings as in the General Formula (I) of claim 1 ; and

reacting the compound represented by the General Formula (IV) with pyrroline-5-carboxylate reductase to produce hydroxy-L-pipecolic acid represented by the following General Formula (III):

wherein R 1 , R 2 , and R 3 have the same meanings as in the General Formula (I) of claim 1 .

8. A method for producing hydroxy-L-pipecolic acid, said method comprising:

producing hydroxy-L-lysine by the method of claim 1 ; and

reacting the collected hydroxy-L-lysine with lysine cyclodeaminase to produce hydroxy-L-pipecolic acid represented by the following General Formula (III):

wherein R 1 , R 2 , and R 3 have the same meanings as in the General Formula (I) of claim 1 .

9. The method for producing hydroxy-L-lysine according to claim 1 , wherein said 2-oxoglutarate-dependent L-lysine hydroxylase comprises a polypeptide selected from the group consisting of:

(A) a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or 12;

(B) a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or 12 except that at least one and not more than 10 amino acids are deleted substituted, and/or added, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity; and

(C) a polypeptide comprising an amino acid sequence with a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:4 or 12, wherein the polypeptide has 2-oxoglutarate-dependent L-lysine hydroxylase activity.