Method for producing hydroxy-L-pipecolic acid
A novel method of producing high-purity hydroxy-L-pipecolic acids in an efficient and inexpensive manner while suppressing the production of hydroxy-L-proline is provided. The method includes allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion, wherein the L-pipecolic acid hydroxylase has the properties: (1) the enzyme can act on L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ion to add a hydroxy group to the carbon atom at positions 3, 4, and/or 5 of L-pipecolic acid; and (2) the enzyme has a catalytic efficiency (kcat/Km) with L-proline that is equal to or less than 7 times the catalytic efficiency (kcat/Km) with L-pipecolic acid.
1. L-pipecolic acid hydroxylase, comprising a protein consisting of an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, the protein has one or more amino acid substitution with other amino acid selected from: substitution of phenylalanine at position 5, substitution of cysteine at position 23, and substitution of aspartic acid at position 282.
2. The L-pipecolic acid hydroxylase according to claim 1 , wherein the protein has one or more amino acid substitution selected from: substitution of phenylalanine with tyrosine at position 5, substitution of cysteine with alanine at position 23, and substitution of aspartic acid with glutamic acid at position 282.
3. The L-pipecolic acid hydroxylase according to claim 1 , wherein DNA encoding the L-pipecolic acid hydroxylase comprises the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1 except that one or several bases are deleted, substituted, inserted, and/or added, which DNA encodes a protein consisting of an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, the protein has one or more amino acid substitution with other amino acid selected from: substitution of phenylalanine at position 5, substitution of cysteine at position 23, and substitution of aspartic acid at position 282.
4. A method of producing a hydroxy-L-pipecolic acid, the method comprising:
allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion,
wherein the L-pipecolic acid hydroxylase comprises a protein consisting of an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, the protein has one or more amino acid substitution with other amino acid selected from: substitution of phenylalanine at position 5, substitution of cysteine at position 23, and substitution of aspartic acid at position 282.
5. The method of producing a hydroxy-L-pipecolic acid according to claim 4 ,
wherein the protein has one or more amino acid substitution selected from: substitution of phenylalanine with tyrosine at position 5, substitution of cysteine with alanine at position 23, and substitution of aspartic acid with glutamic acid at position 282.
6. The method of producing a hydroxy-L-pipecolic acid according to claim 4 , further comprising:
allowing L-lysine and/or DL-lysine to react with
(i-1) one or more enzymes selected from the group consisting of an L-amino acid oxidase, an L-amino acid dehydrogenase and an L-amino acid aminotransferase, or
(i-2) an amino acid racemase and one or more enzymes selected from the group consisting of a D-amino acid oxidase, a D-amino acid dehydrogenase and a D-amino acid aminotransferase,
for the production of 3,4,5,6-tetrahydropyridine-2-carboxylic acid; and subsequently
allowing an N-methyl-L-amino acid dehydrogenase to act on the 3,4,5,6-tetrahydropyridine-2-carboxylic acid for the production of the L-pipecolic acid as the substrate.
7. The method of producing a hydroxy-L-pipecolic acid according to claim 4 , further comprising:
allowing L-lysine to react with one or more enzymes selected from the group consisting of an L-lysine 6-oxidase, an L-lysine 6-dehydrogenase and an L-lysine 6-aminotransferase, for the production of 2,3,4,5-tetrahydropyridine-2-carboxylic acid; and subsequently
allowing a pyrroline-5-carboxylate reductase to act on the 2,3,4,5-tetrahydropyridine-2-carboxylic acid for the production of the L-pipecolic acid as the substrate.
8. The method of producing a hydroxy-L-pipecolic acid according to claim 4 , further comprising allowing a lysine cyclodeaminase to act on L-lysine for the production of the L-pipecolic acid as the substrate.
9. The method of producing a hydroxy-L-pipecolic acid according to claim 4 , wherein the content of L-proline in the L-pipecolic acid as the substrate is not more than 10% (w/w).
10. The method of producing a hydroxy-L-pipecolic acid according to claim 4 , wherein the microorganism or cell having the ability to produce the enzyme is a microorganism or cell transformed with DNA encoding the L-pipecolic acid hydroxylase comprises DNA comprising the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1 except that one or several bases are deleted, substituted, inserted, and/or added, which DNA encodes a protein consisting of an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, the protein has one or more amino acid substitution with other amino acid selected from: substitution of phenylalanine at position 5, substitution of cysteine at position 23, and substitution of aspartic acid at position 282.
11. A method of producing a (5S)-hydroxy-L-pipecolic acid, the method comprising:
allowing an L-pipecolic acid hydroxylase, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid comprising the enzyme and obtained by culturing the microorganism or cell, to act on L-pipecolic acid as a substrate in the presence of 2-oxoglutaric acid and ferrous ion,
wherein the L-pipecolic acid hydroxylase comprises a protein consisting of an amino acid sequence with an identity of not less than 90% to the amino acid sequence of SEQ ID NO: 2, the protein has one or more amino acid substitution with other amino acid selected from: substitution of phenylalanine at position 5, substitution of cysteine at position 23, and substitution of aspartic acid at position 282.
12. The method of producing a (5S)-hydroxy-L-pipecolic acid according to claim 11 , wherein the protein has one or more amino acid substitution selected from: substitution of phenylalanine with tyrosine at position 5, substitution of cysteine with alanine at position 23, and substitution of aspartic acid with glutamic acid at position 282.