Mutant l-pipecolic acid hydroxylase and cis-5-hydroxy-l-pipecolic acid production method utilizing same

Patent metadata

Field	Value
Publication number	US-2024043887-A1
Application number	US-202118268788-A
Country	US
Kind code	A1
Filing date	Dec 24, 2021
Priority date	Dec 25, 2020
Publication date	Feb 8, 2024
Grant date	—

Abstract

The present invention aims to provide a mutant L-pipecolic acid hydroxylase for highly productive and highly selective production of cis-5-hydroxy-L-pipecolic acid by hydroxylation of L-pipecolic acid, and to provide a novel method for industrially producing cis-5-hydroxy-L-pipecolic acid from L-pipecolic acid with high productivity at low cost. The present invention provides a mutant L-pipecolic acid hydroxylase having an amino acid sequence that a particular amino acid mutation(s) is/are introduced in the amino acid sequence of SEQ ID NO:2.

First claim

1 . A mutant L-pipecolic acid hydroxylase having an amino acid sequence that at least one amino acid mutation selected from the group consisting of the following (a) to (o) is introduced in the amino acid sequence of SEQ ID NO:2:
(a) amino acid mutation in which the glutamic acid at position 15 is substituted by alanine; (b) amino acid mutation in which the isoleucine at position 28 is substituted by proline; (c) amino acid mutation in which the isoleucine at position 28 is substituted by arginine; (d) amino acid mutation in which the valine at position 31 is substituted by glutamic acid; (e) amino acid mutation in which the cysteine at position 76 is substituted by tyrosine; (f) amino acid mutation in which the isoleucine at position 108 is substituted by arginine; (g) amino acid mutation in which the leucine at position 142 is substituted by arginine; (h) amino acid mutation in which the leucine at position 142 is substituted by lysine; (i) amino acid mutation in which the leucine at position 142 is substituted by asparagine; (j) amino acid mutation in which the leucine at position 142 is substituted by glutamine; (k) amino acid mutation in which the leucine at position 142 is substituted by histidine; (l) amino acid mutation in which the glutamine at position 202 is substituted by proline; (m) amino acid mutation in which the alanine at position 239 is substituted by aspartic acid; (n) amino acid mutation in which the phenylalanine at position 246 is substituted by tyrosine; and (o) amino acid mutation in which the isoleucine at position 271 is substituted by glycine.

2 . The mutant L-pipecolic acid hydroxylase according to claim 1 , having an amino acid sequence that at least two amino acid mutations selected from the group consisting of the (a) to (o) are introduced in the amino acid sequence of SEQ ID NO:2.

3 . The mutant L-pipecolic acid hydroxylase according to claim 1 , having an amino acid sequence that at least three amino acid mutations selected from the group consisting of the (a) to (o) are introduced in the amino acid sequence of SEQ ID NO:2.

4 . A method of producing cis-5-hydroxy-L-pipecolic acid, the method comprising bringing the mutant L-pipecolic acid hydroxylase according to claim 1 , a microorganism or cell having a capacity to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid containing the enzyme obtained by culturing the microorganism or cell, into contact with L-pipecolic acid, to produce cis-5-hydroxy-L-pipecolic acid.

5 . The method of producing cis-5-hydroxy-L-pipecolic acid according to claim 4 , comprising bringing the mutant L-pipecolic acid hydroxylase, a microorganism or cell having a capacity to produce the enzyme, a processed product of the microorganism or cell, and/or a culture liquid containing the enzyme obtained by culturing the microorganism or cell, into contact with L-pipecolic acid in the presence of 2-oxoglutaric acid and ferrous ions.

Assignees

• Api Corp

Inventors

• Asano Yasuhisa
• Shinoda Suguru
• Enoki Junichi
• Miyake Ryoma
• Sasano Haruka

Classifications

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12Y114/11002

  Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase · CPC title

• C12R2001/19

  Escherichia coli · CPC title