Technical field
[0001]
　The present invention relates to a manufacturing method of cis-5-hydroxy -L- pipecolic acid using an enzyme having the ability to produce cis-5-hydroxy -L- pipecolic acid.
Background technique
[0002]
　Cis-5-hydroxy -L- pipecolic acid (hereinafter sometimes referred to as "5OH-PA".) Is a useful compound as an intermediate or the like of pharmaceuticals. Cis-5-hydroxy -L- pipecolic acid is known to be produced by biological methods from L- pipecolic acid.
[0003]
　Lotus rhizobia Mesorhizobium loti (Mesorhizobium loti) MAFF303099 BAB52605 proteins and alfalfa rhizobia Sinorhizobium Merirochi (Sinorhizobiummeli loti) from 1021 derived CAC47686 protein (hereinafter sometimes referred to as "SmPH".) Is cis L- proline to have the ability to convert 4-hydroxy-proline has been reported (Patent Document 1).
[0004]
　BAB52605 protein, L- pipecolic acid has the ability to convert cis-5-hydroxy -L- pipecolic acid, its productivity has been reported that relatively low (Patent Document 2).
[0005]
　CAC47686 proteins have been reported to have an ability to convert L- pipecolic acid cis-5-hydroxy -L- pipecolic acid from L- pipecolic acid, with cis-5-hydroxy -L- pipecolic acid , (sometimes hereinafter referred to as "3OH-PA".) cis-3-hydroxy-pipecolic acid is also intended to about the same amount produced (Patent Document 2).
[0006]
　Further, Patent Document 2, Seguniriparasu-Rugosasu (Segniliparus rugosus) ATCC BAA-974 derived EFV12517 protein 48 bases (16 equivalent amino acids) than annotation polynucleotide expressed from the upstream (cis gene) in the encoded protein (sometimes hereinafter referred to as "SruPH".) E. coli expressing it, has a cis -5-position hydroxylase activity of L- pipecolic acid, hydroxy-cis-5-L- pipecolic acid -L- it has been reported that can be converted into pipecolic acid. However, as shown in the examples herein, this protein, the L- pipecolic acid, with cis-5-hydroxy -L- pipecolic acid, intended to produce about 2% of cis-3-hydroxy-pipecolic acid is there.
[0007]
　Patent Document 3, L-a pipecolic acid, a method for producing cis-5-hydroxy -L- pipecolic acid have been reported. In Patent Document 3, although modifying the gene SmPH in order to suppress the production of unwanted cis-3-hydroxy pipecolic acid, cis-3-hydroxy-pipecolic acid is produced about 9% be modified it has been reported that.
[0008]
　Any Non-Patent Document 1, it has been reported that the production of cis-5-hydroxy -L- pipecolic acid and cis-3-hydroxy-pipecolic acid SmPH from L- pipecolic acid. In Non-Patent Document 1, although modified SmPH genes in order to increase the productivity of cis-5-hydroxy -L- pipecolic acid, it is modified cis-3-hydroxy pipecolic acid produced 4% Rukoto have been reported.
CITATION
Patent Literature
[0009]
Patent Document 1: WO2009 / 139365
Patent Document 2: WO2013 / 187438
Patent Document 3: WO2013 / 169725
Non-Patent Document
[0010]
Non-Patent Document 1: Koketsu et, ACS Synth Biol, DOI:. 10.1021 / sb500247a
Summary of the Invention
Problems that the Invention is to Solve
[0011]
　Thus, L- from pipecolic acid, by biological methods although the process for producing a cis-5-hydroxy -L- pipecolic acid known, any of cis-5-hydroxy -L- pipecolic acid productivity is low, and also, to a certain extent producing cis-3-hydroxy -L- pipecolic acid. Therefore, as the method for preparing intermediate or the like of pharmaceuticals high purity is required, more efficient method for producing high-purity cis-5-hydroxy -L- pipecolic acid has been desired.
[0012]
　The present invention aims to produce cis-3-hydroxy -L- pipecolic acid is small, more efficient, provides a novel method for producing a high optical purity of cis-5-hydroxy -L- pipecolic acid to.
Means for Solving the Problems
[0013]
　The present inventors have made intensive studies in order to solve the above problems, (sometimes hereinafter referred to as "XdPH".) Kisenorabudasu · Dousetie (Xenorhabdus doucetiae) FRM16 strain derived proteins and Kisenorabudasu-Romani (Xenorhabdus Romanii) str. Pr06-A line-derived protein (hereinafter sometimes referred to as "XrPH".) Has been found to have a high L- pipecolic cis -5-position hydroxylase activity of acid. Then, using the DNA encoding these proteins to prepare a transformant, the transformant cells, by acting on the preparation and / or culture L- pipecolic acid, high optical purity and high concentration in, we found that it is possible to produce cis-5-hydroxy -L- pipecolic acid. The present invention has been completed based on these findings.
[0014]
　That is, the gist of the present invention is as follows.
　[1] L- to pipecolic acid, 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, microorganisms or cells, processed product of the microorganism or cells capable of producing the enzyme, and / or the microorganism or cell It was allowed to act on medium containing the enzyme obtained by culturing, characterized in that to produce cis-5-hydroxy -L- pipecolic acid production method of cis-5-hydroxy -L- pipecolic acid a is,
　the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, comprising the following (a), a polypeptide shown in (B) or (C), cis-5- hydroxy -L- pipecolic acid manufacturing method of:
　(a) SEQ ID NO: 4, or a polypeptide having an amino acid sequence represented by 11;
　(B) SEQ ID NO: 4, or an amino acid sequence represented by 11, one or several amino acids Or; but deletions, substituted and / or added in the amino acid sequence, a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity
　(C) represented by SEQ ID NO: 4 or 11, It has an amino acid sequence having the amino acid sequence 60% or more identity, and a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity.
　[2] The 2-oxoglutarate-dependent L- pipecolic encoding acid hydroxylase DNA is less than (D), comprising a DNA shown in (E) or (F), cis-5 according to [1] - method for producing hydroxy -L- pipecolic acid:
　(D) SEQ ID NO: 1,9, or DNA having the nucleotide sequence represented by 10;
　(E) SEQ ID NO: 1,9, or one or several nucleotide substitutions in the nucleotide sequence represented by 10, comprises a deletion and / or added in the nucleotide sequence, and 2-oxoglutarate-dependent L- pipecolic DNA encoding a polypeptide having an acid hydroxylase activity; or
　comprises a nucleotide sequence that hybridizes under complementary strand under stringent conditions the nucleotide sequence represented by (F) SEQ ID NO: 1, 9, or 10, and DNA encoding a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity.
　[3] 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, obtained by culturing microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or the microorganism or cell Media containing the enzyme was in the presence of 2-oxoglutarate and divalent iron ions, to act on the L- pipecolic acid, cis-5-hydroxy -L- according to [1] or [2] manufacturing method of pipecolic acid.
　[4] acts on L- pipecolic acid has an activity of producing cis-5-hydroxy -L- pipecolic acid, and, following (A), comprising a polypeptide as shown in (B) or (C) , 2-oxoglutarate-dependent L- pipecolic acid hydroxylase protein:
　polypeptide having an amino acid sequence represented by (a) SEQ ID NO: 4 or 11;
　in (B) the amino acid sequence represented by SEQ ID NO: 4 or 11 , one or several amino acids has deletion, substitution and / or added in the amino acid sequence, a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity; or
　(C) SEQ ID NO: 4 or it has an amino acid sequence having the amino acid sequence 60% or more identity represented by 11, and a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity.
　[5] The polypeptide having the amino acid sequence represented by SEQ ID NO: 11.
Effect of the Invention
[0015]
　According to the present invention, L- from pipecolic acid, it can be produced more efficiently with high optical purity of cis-5-hydroxy -L- pipecolic acid. Furthermore, since cis-3 generation of hydroxy -L- pipecolic acid is small, in industrial scale production, at low cost, it is possible to produce a high-purity cis-5-hydroxy -L- pipecolic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016]
Is a diagram illustrating a FIG. 1 various L- pipecolic acid hydroxylase gene introduced in E. coli SDS- polyacrylamide electrophoresis results (electrophoresis photograph). Arrow indicates the band of L- pipecolic acid hydroxylase.
It is a schematic drawing illustrating the analytic results of [2] Various L- pipecolic acid hydroxylase gene reaction product from the introduced E. coli.
3 is a diagram showing a temperature dependence of the various L- pipecolic acid hydroxylase activity.
DESCRIPTION OF THE INVENTION
[0017]
　Hereinafter, the present invention will be described in detail.
[0018]
　In this specification, the term "L- pipecolic acid ability to convert cis-5-hydroxy -L- pipecolic acid", 2-oxoglutarate-dependent manner 5-position hydroxy group to a carbon atom of L- pipecolic acid It refers to the ability to be added.
[0019]
　Whether it has a "ability to convert L- pipecolic acid cis-5-hydroxy -L- pipecolic acid", for example, L- pipecolic acid containing as a substrate, further contains a 2-oxoglutarate as a coenzyme confirmed in reaction system, L- to pipecolic acid, by the action of an enzyme of interest measured by directly measuring the amount of converted cis-5-hydroxy -L- pipecolic acid from L- pipecolic acid can do.
[0020]
　Further, the "enzyme" herein, (. Containing partially purified enzyme) purified enzyme or those immobilized using known immobilization techniques, such as polyacrylamide, carriers such as carrageenan gel those that have been immobilized on the like are also included.
[0021]
　As used herein, "L- pipecolic acid microorganism or cells having an ability to convert cis-5-hydroxy -L- pipecolic acid" (hereinafter, may be referred to as "microorganism or cell of the present invention".) And is particularly limited as long as it has a "L- pipecolic 5-position of the ability which can be added to hydroxy groups to carbon atoms of the acid" is not, it may be a microorganism or cells having an inherently the ability, it may be a microorganism or cells conferred the ability by breeding. As means for imparting the ability by breeding, including genetic engineering processes (transformation) and mutagenesis, it is possible to employ a known method. As the method of transformation, it is possible to use a method such as introducing a gene of interest, to enhance expression of a gene of interest, such as by modifying an expression regulatory sequence such as a promoter on the chromosome.
[0022]
　As the type of "microbial or cell" include those described in a host organism or host cell to be described later. Further, in the present specification, the "L- to 5-position carbon atoms of the pipecolic acid microorganism or cells having an ability which can be added the hydroxy group" is not limited to the microorganisms or cells alive, as the biological dead It is but also include those having an enzymatic capability.
[0023]
　In the present specification, the type of organism "host organism" is not particularly limited, Escherichia coli, Bacillus subtilis, coryneform bacteria, bacteria belonging to the genus Pseudomonas, Bacillus bacteria, Rhizobium bacteria, lactobacilli, succinonitrile Bacillus bacteria, Ana erotic bios pyridinium ram bacteria, Actinobacillus bacteria such prokaryotes, yeasts, fungi such as filamentous fungi, plant, include eukaryotes such as animals. Among them, preferably, Escherichia coli, yeast, a coryneform bacterium, particularly preferably E. coli.
[0024]
　In the present specification, the type of cells that "host cell" is not particularly limited, it is possible to use animal cells, plant cells, insect cells and the like.
[0025]
　As used herein, "expression vector", by introducing into the host organism incorporate polynucleotide encoding a protein having the desired function, in order to replicate and express a protein having a desired function in said host organism is a genetic factor to be used. For example, a plasmid, virus, phage, but cosmids and the like without limitation. Preferably, the expression vector is a plasmid.
[0026]
　As used herein, "transformant", the gene of interest by using a said expression vector has been introduced, microorganisms can now be expressed desired traits associated with proteins having a desired function or cell means.
[0027]
　As used herein, "treatment of a microorganism or cell", culturing microorganisms or cells, the microorganisms or cells, 1) those treated with an organic solvent or the like, 2) a lyophilized, 3) a carrier, such as which was immobilized, 4) are those physically or enzymatically disrupted, and means such as those containing a protein having the desired function.
[0028]
　In the present specification, "culture solution containing enzyme obtained by culturing a microorganism or cell", 1) the culture medium of the microorganism or cell, 2) was treated with an organic solvent such as a culture broth of a microorganism or cell culture, 3) means a culture medium are physically or enzymatically disrupt the cell membrane of the microorganism or cell.
[0029]
<2-oxoglutarate-dependent L- pipecolic acid production method of cis-5-hydroxy -L- pipecolic acid using the hydroxylase>
　method for producing a cis-5-hydroxy -L- pipecolic acid of the present invention, L - the pipecolic acid, 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or by culturing a microorganism or cell exerting a culture solution containing the obtained enzyme characterized. Production method of the present invention, as described later, the presence of 2-oxoglutarate and divalent iron ions, is preferably performed.
[0030]
　2-oxoglutarate-dependent L- pipecolic acid hydroxylase are used in the present invention (hereinafter sometimes referred to as "L- pipecolic acid hydroxylase of the present invention".) Is, hydroxide L- pipecolic acid because of the high regioselectivity and stereoselectivity when, by using this efficiently, it is possible to obtain a high optical purity of cis-5-hydroxy -L- pipecolic acid.
[0031]
　2-oxoglutarate-dependent L- pipecolic acid hydroxylase of the present invention is not particularly limited as long as it is an enzyme capable of converting the L- pipecolic acid cis-5-hydroxy -L- pipecolic acid, but the sequence having the amino acid sequence set forth in ID NO: 4 or 11, or is preferably a homologue of the amino acid sequence and has a 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity. That, L- pipecolic acid hydroxylase of the present invention, the following (A), is preferably one comprising a polypeptide as shown in (B) or (C).
　Polypeptide having the amino acid sequence represented by (A) SEQ ID NO: 4 or 11;
　in the amino acid sequence represented by (B) SEQ ID NO: 4 or 11, one or several amino acids are deleted, substituted and / or added or; has the amino acid sequence, 2-oxoglutarate-dependent L- polypeptide having pipecolic acid hydroxylation activity
　having (C) an amino acid sequence at least 60% identity with SEQ ID NO: 4 or 11 It has an amino acid sequence, and polypeptides having 2-oxoglutarate-dependent L- pipecolic acid hydroxylation activity.
[0032]
　Can be used in the present invention, the homolog of 2-oxoglutarate-dependent L- pipecolic acid hydroxylase having the amino acid sequence set forth in SEQ ID NO: 4 or 11, as described in the (B), 2-oxoglutarate long as it retains the dependent L- pipecolic acid hydroxylating activity, in the amino acid sequence set forth in SEQ ID NO: 4 or 11, one or several amino acids are deleted, include those having a substituent, or added in the amino acid sequence . Here, "one or several amino acids" is, for example, 1 to 100, preferably from 1 to 50, more preferably 1 to 20, 1 to 10 more preferably, particularly preferably it is a 1 to 5 amino acids.
[0033]
　Also, the homologues as described in (C), 2-oxoglutarate long as it retains the acid-dependent L- pipecolic acid hydroxylation, entire amino acid sequence shown in SEQ ID NO: 4 or 11 and at least 60% or more, preferably 80% or more, more preferably 90% or more, more preferably 95%, particularly preferably may be a protein with a sequence identity of 99% or more.
[0034]
　Amino acid sequence set forth in SEQ ID NO: 4 is based on Kisenorabudasu-Dousetie (Xenorhabdus doucetiae) FRM16 strain known genomic information.
　The amino acid sequence of SEQ ID NO: 11, Kisenorabudasu-Romani (Xenorhabdus romanii) str. It is based on cloned gene information by a known method using PCR from Pr06-A strain.
[0035]
　Amino acid sequence of SEQ ID NO: 4 or 11, and L- pipecolic acid hydroxylase of the present invention including a homologue thereof, so selectively hydroxylate 5-position carbon atoms of the L- pipecolic acid, cis with high efficiency - it is possible to produce a 5-hydroxy -L- pipecolic acid.
[0036]
　In the production method of the present invention may be used in combination with a plurality of 2-oxoglutarate-dependent L- pipecolic acid hydroxylase.
[0037]
　May be used in the present invention 2-oxoglutarate-dependent L- pipecolic acid hydroxylase is Kisenorabudasu-Dousetie (Xenorhabdus doucetiae) FRM16 strain or Kisenorabudasu-Romani (Xenorhabdus romanii) str. Can also be obtained by purification from Pr06-A strain, the DNA encoding the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase cloned the gene by a known method such as PCR or hybridization, it It can also be obtained by expressing in a suitable host.
[0038]
　As the DNA encoding the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase having the amino acid sequence shown in SEQ ID NO: 4 or 11, respectively, include a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 9 or 10 , 2-oxoglutarate-dependent L- pipecolic long as it encodes a protein having an acid hydroxylation activity, or a homologue of a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 9 or 10. That is, the DNA encoding the L- pipecolic acid hydroxylase of the present invention, the following (D), include the nucleotide sequence shown in (E) or (F).
　(D) DNA having the nucleotide sequence represented by SEQ ID NO: 1, 9, or 10;
　(E) 1 or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 1, 9, or 10 substitutions, deletions, and / or wherein the added nucleotide sequence, and 2-oxoglutarate-dependent L- pipecolic encoding a polypeptide having an acid hydroxide active DNA; or
　(F) of the base sequence represented by SEQ ID NO: 1, 9 or 10 DNA encoding a polypeptide having a complementary strand comprises hybridizing nucleotide sequence under stringent conditions, and 2-oxoglutarate-dependent L- pipecolic acid hydroxylation activity.
[0039]
　The homologues, for example, as described in the (E), 1 or several nucleotides in the nucleotide sequence of SEQ ID NO: 1, 9 or 10 are substituted, include those containing a deletion or addition of nucleotide sequence. And one or several bases here, for example, 1 to 300, preferably 1 to 150, more preferably 1 to 60, more preferably 1 to 30, particularly preferably 1 number to 15, which is the base.
　Note that SEQ ID NO: 1 is a gene Kisenorabudasu-Dousetie (Xenorhabdus doucetiae) FRM16 strain which encodes the amino acid sequence of SEQ ID NO: 4 a nucleotide sequence with optimized codons for E. coli expression. This is included in the DNA codon encoding optimized DNA may also be used of course the present invention 2-oxoglutarate-dependent L- pipecolic acid hydroxylase depending on the host transformed target as.
[0040]
　Furthermore, homologues of the DNA, the as described in (F), 2-oxoglutarate-dependent L- pipecolic long as it encodes a protein having an acid hydroxylation activity, complementary nucleotide sequence of SEQ ID NO: 1, 9 or 10 a chain under stringent conditions may be a hybridizing DNA. Here, the hybridizing nucleotide sequence "under stringent conditions, DNA used as a probe is used under stringent conditions, colony hybridization, plaque hybridization, or Southern blot hybridization method or the like It means a nucleotide sequence of the obtained DNA by. Stringent conditions, for example, in a colony hybridization and plaque hybridization, using a filter immobilized with DNA or fragment of the DNA derived from a colony or plaque, 0.7 mol / L ~ 1.0 mol / presence of aqueous sodium chloride solution L, after performing hybridization at 65 ° C., the composition of 0.1 ~ 2 × SSC solution (1 × SSC, 150mmol / L sodium chloride aqueous solution, 15 mmol / L sodium citrate solution ) was used, mention may be made of the conditions for washing the filter under conditions of 65 ° C..
　Each hybridization, Molecular Cloning: A laboratory Mannual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989. It can be carried out according to the method described in equal.
[0041]
　Those skilled in the art, SEQ ID NO: 1, 9 or 10 DNA to site-directed mutagenesis of (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)) and the like optionally substituted with, deletions, by introducing inserts and / or addition mutation, it is possible to obtain a DNA homologs described above .
[0042]
　See also SEQ ID NO: 4 or 11 amino acid sequence or a portion thereof and, based on the base sequence or a portion thereof represented by SEQ ID NO: 1, 9 or 10, for example DNA Databank of JAPAN (DDBJ), etc. Database performing homology search against, it is also possible to get a nucleotide sequence information of the DNA amino information or encoding the same 2-oxoglutarate-dependent L- pipecolic acid hydroxylase.
[0043]
　In the method for producing hydroxy -L- pipecolic acid of the present invention, 2-oxoglutarate-dependent L- pipecolic may be used directly to the reaction of the acid hydroxylase, but 2-oxoglutarate-dependent L- pipecolic acid solution microorganisms or cells comprising the oxidase, processed product of the microorganism or cells, and / or it is preferable to use a culture solution containing the enzyme obtained by culturing the microorganism or cell.
[0044]
　2-The-oxoglutarate-dependent L- pipecolic microorganism or cell containing an acid hydroxylase, originally 2-oxoglutarate-dependent L- pipecolic may be used a microorganism or cells having an acid hydroxylase but, 2- oxoglutarate it is preferred to use a microorganism or cell transformed with the gene coding for acid-dependent L- pipecolic acid hydroxylase.
[0045]
　Further, 2-The-oxoglutarate-dependent L- pipecolic preparation of a microorganism or cell containing an acid hydroxylase, for example, microorganism or cell with acetone, dimethyl sulfoxide (DMSO), an organic solvent or a surfactant such as toluene those treated by freeze drying the treated ones, those taking out preparation of physically or enzymatically disrupted microbial or cell such as those, the enzyme fraction of the microorganism or in the cell as a crude product or a purified product, more , these can be used polyacrylamide gel, such as those immobilized on a carrier represented by carrageenan gel or the like.
[0046]
　The 2-oxoglutarate-dependent L- pipecolic acid culture solution obtained by culturing a microorganism or cell containing a hydroxylase, for example, a suspension or of the microorganism or cell and the liquid medium, the microorganism or cell If a secreted expression type microorganism or cell may use supernatant or the concentrate with the microorganism or cell is removed by centrifugation.
[0047]
　It was isolated as described above, by inserting an expressible form DNA known expression vector encoding the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, 2-oxoglutarate-dependent L - pipecolic acid hydroxylase expression vector is provided. Then, by transforming a host cell with this expression vector can be a DNA encoding a 2-oxoglutarate-dependent L- pipecolic acid hydroxylase obtain a transformant introduced. Transformants can also be obtained by incorporating in an expressible state by a technique such as DNA homologous recombination that host encoding chromosomal DNA in 2-oxoglutarate-dependent L- pipecolic acid hydroxylase of.
[0048]
　As a manufacturing method of the transformant, specifically, the code in the host cell a plasmid vector or phage vector or viral vector that is stable in the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase such microorganisms the a DNA was introduced, or introducing an expression vector constructed in host cells, or by introducing the DNA directly into the host genome, methods for the genetic information transcribed and translated and the like. In this case, preferably it is linked to the 5'-side upstream of the DNA to a suitable promoter in a host, further it is more preferable to connect the terminator to the 3'-downstream. Such promoters and terminators are not particularly limited as long as promoters and terminators are known to function in cells used as host, for example, "Fundamental Course in Microbiology (8): Genetic Engineering", Kyoritsu Shuppan, etc. vectors available in the host microorganism, promoter and terminator are described in detail.
[0049]
　2 The host microorganism to be transformed for oxoglutarate express an acid dependent L- pipecolic acid hydroxylase, the host itself is particularly limited as long as it does not adversely affect the reaction of L- pipecolic acid rather, it can be specifically mentioned microorganisms as described below.
[0050]
　Escherichia (Escherichia) genus Bacillus (Bacillus) genus Pseudomonas (Pseudomonas) genus Serratia (Serratia) genus Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus Streptococcus (Streptococcus) genus Lactobacillus (Lactobacillus ) genus host-vector system established by which bacteria belonging to such.
[0051]
　Rhodococcus (Rhodococcus) genus Streptomyces (Streptomyces) genus established by and actinomycetes host vector systems that belong to such. Saccharomyces (Saccharomyces) genus Kluyveromyces (Kluyveromyces) sp., Schizosaccharomyces (Schizosaccharomyces) sp., Zygosaccharomyces (Zygosaccharomyces) genus Yarrowia (Yarrowia) genus Trichosporon (Trichosporon) genus, Rhodosporidium (Rhodosporidium) genus, Hansenula (Hansenula) spp, Pichia (Pichia) sp., Candida (Candida) spp established in which yeast host vector systems that belong to such.
[0052]
　Neurospora (Neurospora) genus Aspergillus (Aspergillus) genus Cephalosporium (Cephalosporium) genus Trichoderma (Trichoderma) sp established by that mold of host vector systems that belong to such.
[0053]
　Procedure for the transformant prepared, how to build and host cultures of recombinant vector compatible with the host are molecular biology can be carried out according to the technique which is commonly biotechnology, in the field of genetic engineering ( for example, the method described in Molecular Cloning).
[0054]
　Hereinafter, specifically, the preferred host microorganisms, the method of the preferred transformation in each organism, vector, promoter, examples of such terminators, the present invention is not limited to these examples.
[0055]
　Escherichia, in particular Escherichia coli (Escherichia coli), as a plasmid vector, pBR, etc. pUC series plasmids can be mentioned, lac (beta-galactosidase), trp (tryptophan operon), tac, trc (lac, the trp fusion), λ phage PL, such as a promoter derived from such as PR, and the like. As the terminator, origin trpA, phage-derived, and the like rrnB ribosomal RNA.
[0056]
　In Bacillus, vectors, pUB110 based plasmid, and the like can be illustrated pC194 series plasmids, and can be integrated into host chromosome. The promoter and terminator, alkaline protease, neutral protease, such as promoters and terminators gene of α- amylase or the like can be used.
[0057]
　In genus Pseudomonas, vectors, Pseudomonas putida (Pseudomonas putida), and a common host vector system has been established, etc. Pseudomonas cepacia (Pseudomonas cepacia), plasmids responsible for the degradation of toluene compounds, the TOL plasmid broad host range vector to the base (including genes required for autonomous replication such as from RSF1010) pKT240 (gene, 26,273-82 (1983)) and the like.
[0058]
　Brevibacterium, in particular in Brevibacterium lactofermentum (Brevibacterium lactofermentum), as the vector include plasmid vectors such as pAJ43 (Gene 39,281 (1985)). The promoter and terminator, various promoters and terminators used in Escherichia coli is available.
[0059]
　The genus Corynebacterium, especially in Corynebacterium glutamicum (Corynebacterium glutamicum), as the vector, (JP-A-57-183799) pCS11, pCB101 (Mol.Gen.Genet.196,175 (1984)), etc. like plasmid vector of.
[0060]
　Saccharomyces (Saccharomyces) genus, particularly in Saccharomyces cerevisiae (Saccharomyces cerevisiae), as a vector, YRp series, YEp series, YCp series, and a YIp series plasmids. Further, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, beta-galactosidase, phosphoglycerate kinase, the promoters of various enzyme genes such as enolase, terminator are available.
[0061]
　Schizosaccharomyces in Streptomyces (Schizosaccharomyces) genus, vectors, Mol. Cell. Biol. , And the like Schizosaccharomyces plasmid vectors derived from Streptomyces pombe according to 6,80 (1986). In particular, PAUR224 are readily available are commercially available from Takara Bio Inc..
[0062]
　In Aspergillus (Aspergillus) genus Aspergillus niger (Aspergillus niger), Aspergillus Oriji (Aspergillus oryzae), etc. have been studied among fungi, integration into the plasmid and chromosome are available, bacterial cells promoter derived from outside the protease and amylase are available (Trendsin Biotechnology 7,283-287 (1989)).
[0063]
　Also, other than the above, and host-vector system in accordance with various microorganisms is established, it is possible to them appropriately used.
[0064]
　Further, other than microorganisms, in plants and animals have been established various host-vector systems, in particular insects (e.g., silkworm) in an animal, such as (Nature 315,592-594 (1985)) and, rapeseed, corn, large quantities or system for expressing heterologous proteins in plants, such as potatoes, and a system using cell-free protein synthesis system such as E. coli cell-free extract and wheat germ is established, can be suitably used.
[0065]
　In the production method of the present invention, 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, cultured microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or the microorganism or cell Media containing the enzyme obtained by 2-presence of oxoglutarate, by acting on certain L- pipecolic acid in the reaction substrate, is possible to produce cis-5-hydroxy -L- pipecolic acid it can.
[0066]
　Production method of the present invention, L- a pipecolic acid, 2-oxoglutarate, and 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, microorganisms or cells capable of producing the enzyme, the microorganism or cell treated product, and / or it is not particularly limited as long as it can act on medium containing the enzyme obtained by culturing the microorganism or cell, usually in an aqueous medium, or with the aqueous medium and an organic solvent it is preferably carried out in a mixture. Production method of the present invention preferably further carried out in the presence of divalent iron ions.
[0067]
　As the aqueous medium, e.g., water or buffers.
[0068]
　As examples of the organic solvent, the use of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, alcohol solvents such as tert- butanol, acetone, and has high solubility of the reaction substrate such as dimethyl sulfoxide can. The organic solvent also is effective in removal of the reaction by-products, ethyl acetate, butyl acetate, toluene, chloroform, it can also be used n- hexane.
[0069]
　The reaction substrate L- pipecolic acid is usually 0.01% substrate concentration w / v ~ 90% w / v, and preferably in the range of 0.1% w / v ~ 30% w / v. Reaction substrate, may be added at once at the start of the reaction, from the viewpoint of improving the concentration of accumulated points and product of reducing the effects of a substrate inhibition of the enzyme, continuous or intermittent it is desirable to add to.
[0070]
　2-oxoglutarate is normally substrate and an equimolar or more, preferably added in the range of equimolar to 1.2 times molar. 2-oxoglutarate, which may be added at once at the start of the reaction, from the viewpoint of improving the concentration of accumulated points and product of reducing the effects of a inhibitory effect on the enzyme, continuous or intermittently, it is preferable to add. Or, 2-oxoglutarate host such as glucose instead of acid added metabolizable inexpensive compounds, is metabolized in the host, it is also possible to use in the reaction and the resulting 2-oxoglutarate in the process.
[0071]
　Production method of the present invention is preferably carried out in the presence of divalent iron ions. Divalent iron ions, usually 0.01mmol / L ~ 100mmol / L, preferably is preferably used in a range of 0.1mmol / L ~ 10mmol / L. The divalent iron ion, as such iron sulfate, can be added at once at the start of the reaction, during the reaction, divalent iron ions or are oxidized to trivalent added, to form a precipitate reduction it is also effective to add added or when you got. Also, L- pipecolic acid hydroxylase of the present invention, microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or the enzyme obtained by culturing the microorganism or cell not necessarily added if it contains divalent iron ions already sufficient amount medium containing.
[0072]
　The reaction is generally 4 ° C. ~ 60 ° C., preferably from 15 ° C. ~ 45 ° C., particularly preferably at a reaction temperature of 20 ° C. ~ 40 ° C., usually pH 3 ~ pH 11, preferably at pH 5 ~ pH 8. The reaction time is usually about 1 hour to 72 hours.
[0073]
　Microorganisms or cells are added to the reaction solution, processed product of the microorganism or cells, and / or amount of the culture solution containing the enzyme obtained by culturing the microorganism or cell, for example when adding cells reaction Usually the concentration of the cells, 0.1% w / v ~ 50% w / v of about at wet cells weight, preferably added in an amount of 1% w / v ~ 20% w / v, the treated If used, determine the specific activity of the enzyme is added in an amount such that the cell concentration when added.
[0074]
　Hydroxy -L- pipecolic acid produced by the production method of the present invention, after the completion of the reaction, after separation due to such bacteria and proteins in the reaction mixture centrifuged, membrane treatment, 1-butanol, such as tert- butanol extraction with an organic solvent, distillation, column chromatography using ion exchange resins or silica gel, crystallization or monohydrochloride salt in isoelectric point, purified by combining dihydrochloride, the crystallization and the like of the calcium salt or the like as appropriate It can be carried out.
[0075]
[Example]
　Hereinafter, this invention will be illustrated in detail by Examples, the present invention is not limited thereto.
Example 1
[0076]
2-oxoglutarate-dependent pipecolic acid cloning of hydroxylase gene
　Kisenorabudasu-Dosetie the (Xenorhabdus doucetiae) FRM16 strain derived putative of L- proline cis-4-hydroxylase XdPH (GenBank Accession No. CDG16639, SEQ ID NO: 4) artificially synthesized codon-optimized gene sequence for E. coli expression encoding (Xdph_Ecodon, SEQ ID NO: 1) at DNA2.0 companies, to prepare a plasmid pJ411XdPH inserted in pJExpress411 (DNA2.0).
　Similarly, it was also cloned representative gene showing a known pipecolic acid 5-position hydroxylating activity. Seguniriparasu-Rugosasu (Segniliparus rugosus) NBRC101839 strain derived from L- pipecolic acid cis-5-hydroxylase SruPH (GenBank Accession No. EFV12517, SEQ ID NO: 6) and, Shinomezobiumu-Merirochi (Sinorhizobium meliloti) from 1021 strain L- proline cis 4-hydroxylase SmPH (GenBank Accession No. CAC47686, SEQ ID NO: 5) is a gene sequence that is codon-optimized for E. coli expression encoding, respectively Sruph_Ecodon (SEQ ID NO: 3) Smph_Ecodon (SEQ ID NO: 2) DNA 2 artificially synthesized by 2.0 companies, each pJexpress411 and (DNA2.0) pJex Inserted plasmid pJ411SruPH to Press401, to prepare a PJ401SmPH. Were synthesized Smph_r (SEQ ID NO: 8) and primer Smph_f (SEQ ID NO: 7) for Smph_Ecodon, the plasmid DNA PCR was carried out according to a conventional method as a template by using them, to obtain a DNA fragment of about 1.0kbp . Resulting restriction DNA fragment enzyme NdeI, it was digested with HindIII, NdeI, by ligating according to a conventional method to pET24a (Novagen) was digested with HindIII, and each give a PET24SmPH.
Example 2
[0077]
2-oxoglutarate and acquisition of dependence pipecolic acid hydroxylase gene expression cells confirmed expression level
　then according to a conventional method E. (Escherichia coli) BL21 (DE3) ( Invitrogen) using the plasmid obtained transformed, recombinant E. coli BL21 (DE3) / pJ411XdPH, BL21 (DE3) / pJ411SruPH, and give the BL21 (DE3) / pET24SmPH. To obtain cells expressing the introduced gene, for each recombinant Escherichia coli, using a liquid LB medium containing kanamycin and lac promoter inducer, 4 hours to 6 hours of culture at 28 ° C., even about at 15 ℃ cells were harvested after culturing for 40 hours.
　The resulting recombinant Escherichia coli turbidity OD 630 so of about 10, was suspended in pH7 of 50mmol / L MES (2-Morpholinoethanesulfonic acid) buffer. Perform ultrasonic treatment the suspension 0.5mL on ice, was separated into supernatant and residue by centrifuging at a rotational speed of the subsequent 12,000 rpm. The resulting supernatant and the soluble fraction, the residue was insoluble fraction.
　After the soluble fraction and insoluble fraction obtained was treated according to a conventional method, to confirm protein expression levels expressed by SDS- polyacrylamide electrophoresis. The results are shown in Figure 1.
Example 3
[0078]
2-oxoglutarate dependent pipecolic acid activity confirmation hydroxylase
　5 mmol / L L-pipecolic acid in a plastic tube, 10 mmol / L 2-oxoglutarate, 1 mmol / L L-ascorbic acid, 0.5 mmol / L iron sulfate, Then, the liquid 0.2mL which is added the crude enzyme solution obtained in example 2 at a protein concentration of about 2 mg / mL, was shaken for 1 hour at 30 ° C..
　The reaction products were derivatized by 1-fluoro-2,4-dinitrophenyl- 5-L-alaninamide (FDAA), and then analyzed by UPLC-MS (manufactured by Waters). As a result, as shown in FIG. 2, it was confirmed that produce compounds that match the retention time 5.3 minutes of each crude enzyme solution from the solution after reaction with 5-hydroxy-pipecolic acid standards. Each enzyme solution of the indicated 5-hydroxy pipecolic generating activity (U / g), the protein amount (g) per each calculated 4.1U / g, 8.9U / g, and 3.1U / g. Wherein units (U) represents the ability to produce 1μ mole of substrate per minute.
　Also, from the SmPH reaction product, it was confirmed that the cis-3-hydroxypivalic compound of retention time 6.0 minutes believed Pekorin acid is produced. The result of comparing the percentage of cis-3-hydroxy-pipecolic acid which occupies the area value hydroxy pipecolic acid peaks on the chromatogram the total hydroxy pipecolic acid amount shown in Table 2. During XdPH reaction products have been shown to only 0.17% cis-3-hydroxy-pipecolic acid was not generated.
　Incidentally, analytical conditions of hydroxy pipecolic acid by HPLC are shown in Table 1 below.
Example 4
[0079]
Temperature dependence of the confirmation of the hydroxylation reaction
　to confirm the temperature dependency of the enzymatic reaction, after incubation for 1 hour 30 minutes at various temperatures crude enzyme solution of each hydroxylase, was allowed to react for one hour at 30 ° C. 5-hydroxy-pipecolic acid production amount after the measurement. The results obtained, the highest activity of each enzyme as a relative activity when used as a 100 (%), shown in FIG.
[0080]
[Table 1]

[0081]
[Table 2]

Example 5
[0082]
2-oxoglutarate-dependent pipecolic acid cloning of hydroxylase gene
　Kisenorabudasu-Dosetie (Xenorhabdus doucetiae) FRM16 strain and is related species Kisenorabudasu-Romani (Xenorhabdus romanii) str. Chromosomal DNA from Pr06-A strain as a template, putative L- proline cis-4-hydroxylase XdPH gene (XdPHori: GenBank Accession No. FO704550- XDD1_0936, SEQ ID NO: 9) is a primer to amplify the XdPHfus-f (SEQ ID NO: 12) and XdPHfus-r (SEQ ID NO: 13) was amplified DNA fragment of about 1kbp respectively by polymerase chain reaction (PCR). The resulting two DN fragment was via the use of an In-fusion HD Cloning Kit (manufactured by Takara Bio Inc.), restriction enzymes NcoI, it was inserted in a conventional manner into pQE60 (QIAGEN) digested with HindIII, respectively PQEXdPHori, the pQEXrPHori Obtained.
[0083]
　Transformation according to a conventional method E. (Escherichia coli) JM109 then using the resulting each plasmid was recombinant E. coli JM109 / pQEXdPHori, and to obtain a JM109 / pQEXrPHori. Usual manner to confirm the inserted gene sequences on pQE60, Kisenorabudasu-Romani (Xenorhabdus romanii) str. Pr06-A line-derived amino acid gene sequence of hydroxylase (xrph_ori, SEQ ID NO: 10) was determined and the amino acid sequence (XrPH, SEQ ID NO: 11). XrPH whereas XdPH, had a 99% identity.
[0084]
　To obtain cells expressing the introduced gene, and harvested after overnight culture using a liquid LB medium containing ampicillin for each recombinant Escherichia coli.
　Plastic tube with 20 mmol / L L-pipecolic acid, 15 mmol / L 2-oxoglutarate, 10mmol / L Tris (2- carboxyethyl) phosphine hydrochloride (TCEP), 0.5mmol / L iron sulfate, 10 mmol / L sodium citrate, 1% Nymeen, and the resulting recombinant E. coli JM109 / PQEXdPHori, and JM109 / cells obtained from the culture solution 0.4ml of pQEXrPHori a pH6.5 of 50mmol / L Bis (2-hydroxyethyl ) iminotris (hydroxymethyl) methane buffer in addition the suspended liquid 0.2 mL, and the reaction solution a total volume of 1 mL, which was shaken for 20 hours at 20 ° C..
　The liquid after the reaction, was derivatized with AccQ · Tag (Waters) and analyzed by HPLC under the following conditions conditions were measured and the resulting 5-hydroxy -L- pipecolic acid.
[0085]
　Column: XBridge C18 5μm (2.1 × 150mm ) ( manufactured by Waters Co.)
Eluent A: 10 mmol / L ammonium acetate (pH 5)
Eluent B: methanol (0 ~ 0.5min (0% ⇒1 %), 0 .5 ~ 18min (1% ⇒5% ), 18 ~ 19min (5% ⇒9%), 19 ~ 29.5min (9% ⇒17%), 29.5 ~ 40min (17% ⇒60%), 40 ~ 43min (60%))
flow rate: 0.3 mL / min
detection: fluorescence detector
temperature: 30 ° C.
[0086]
　The measurement results are shown in Table 3. Kisenorabudasu-Dosetie (Xenorhabdus doucetiae) not only hydroxide enzyme XdPH from FRM16 shares, Kisenorabudasu-Lomani (Xenorhabdus romanii) str. For even Pr06-A strain derived hydroxylase, it was confirmed that hold the pipecolic acid 5-position hydroxylating activity.
[0087]
[table 3]

The scope of the claims
[Claim 1]
　The L- pipecolic acid, and cultured 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or the microorganism or cell by the action of the culture solution containing the enzyme was collected using, characterized in that to produce cis-5-hydroxy -L- pipecolic acid, a process for the preparation of cis-5-hydroxy -L- pipecolic acid ,
　the 2-oxoglutarate-dependent L- pipecolic acid hydroxylase, the following (a), (B) or comprising a polypeptide (C), the method for producing a cis-5-hydroxy -L- pipecolic acid :
　(a) SEQ ID NO: 4, or a polypeptide having an amino acid sequence represented by 11;
　(B) SEQ ID NO: 4, or an amino acid sequence represented by 11, one or several amino acids are deleted , Substituted and / or added in the amino acid sequence, 2-oxoglutarate polypeptide having an acid-dependent L- pipecolic acid hydroxylase activity; or
　a (C) SEQ ID NO: 4, or amino acid sequence represented by 11 It has an amino acid sequence having 60% or more identity, and a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity.
[Claim 2]
　The 2-oxoglutarate-dependent L- pipecolic encoding acid hydroxylase DNA is less than (D), comprising a DNA shown in (E) or (F), cis-5- hydroxy of claim 1 - production method of L- pipecolic acid
　; (D) SEQ ID NO: 1,9, or DNA having the nucleotide sequence represented by 10
　one or several in the nucleotide sequence represented by (E) SEQ ID NO: 1, 9, or 10 number of base substitutions, including deletions and / or added in the nucleotide sequence, and 2-oxoglutarate-dependent L-DNA encoding a polypeptide having a pipecolic acid hydroxylase activity; or
　(F) SEQ ID NO: 1 , 9, or comprises a nucleotide sequence that hybridizes under complementary strand under stringent conditions the nucleotide sequence represented by 10, and code a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity DNA to be.
[Claim 3]
　2-oxoglutarate-dependent L- pipecolic acid hydroxylase, microorganisms or cells capable of producing the enzyme, processed product of the microorganism or cells, and / or enzyme obtained by culturing the microorganism or cell Media containing 2-presence of oxoglutarate and divalent iron ions, to act on the L- pipecolic acid, method for producing a cis-5-hydroxy -L- pipecolic acid according to claim 1 or 2 .
[Claim 4]
　It has the activity of producing cis-5-hydroxy -L- pipecolic acid acts on L- pipecolic acid, and comprises the following (A), a polypeptide shown in (B) or (C), 2- oxoglutarate dependent L- pipecolic acid hydroxylase protein:
　(a) SEQ ID NO: 4, or a polypeptide having an amino acid sequence represented by 11;
　(B) SEQ ID NO: 4, or an amino acid sequence represented by 11, has one or more amino acids are deleted, substituted and / or added in the amino acid sequence, a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity; or
　(C) SEQ ID NO: 4, or it has an amino acid sequence having the amino acid sequence 60% or more identity represented by 11, and a polypeptide having 2-oxoglutarate-dependent L- pipecolic acid hydroxylase activity.
[Claim 5]
Polypeptide having the amino acid sequence represented by SEQ ID NO: 11.