Biological method for producing cis-5-hydroxy-L-pipecolic acid

摘要

A method for producing cis-5-hydroxy-L-pipecolic acid is described. A gene recombinant microorganism enabling direct production of cis-5-hydroxy-L-pipecolic acid can be used in the method. Also described is a gene recombinant microorganism. In particular, it is described that a gene recombinant microorganism having DNAs encoding proteins involved in the biosynthesis of L-pipecolic acid and a DNA encoding a protein having the L-pipecolic acid cis-5-hydroxylase activity is cultured in a medium, and cis-5-hydroxy-L-pipecolic acid is collected from the medium.

主权项

1. A method for producing cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, which comprises a step of producing cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate with a microorganism transformed with a heterologous polynucleotide selected from the group consisting of(A)to (F) in an expressible state:
(A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2, (B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate, wherein the stringent conditions comprise performing hybridization at 50° C. in the presence of a saline sodium citrate (SSC) solution of 2-fold concentration and 50% formamide followed by washing at 65° C. with an SSC solution of 0.1-fold concentration; (C) a polynucleotide showing an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate, wherein the polynucleotide comprises a partial sequence encoding an aspartyl/asparaginyl beta-hydroxylase region and a partial sequence encoding an L-proline 3-hydroxylase C-terminal region; (D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25; (E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of 30 or less amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate, wherein the polynucleotide comprises a partial sequence encoding an aspartyl/asparaginyl beta-hydroxylase region and a partial sequence encoding an L-proline 3-hydroxylase C-terminal region; and (F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate, wherein the polynucleotide comprises a partial sequence encoding an aspartyl/asparaginyl beta-hydroxylase region and a partial sequence encoding an L-proline 3-hydroxylase C-terminal region.