JP2011239707A - Method of producing peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing a peptide using a protein having peptide synthetic activity or a transformant for developing the protein, in which the protein synthesizes the peptide with a free amino acid as a substrate.SOLUTION: The method produces the peptide by using the protein described in any of the following proteins [1]-[3], a culture of the transformant for developing the protein, or a processed material of the culture as an enzyme source, or cultivating the transformant for developing the protein: [1] the protein having a specified amino acid sequence; [2] the protein including the amino acid sequence in which one or more amino acid residues are deleted, replaced or added in the amino acid sequence, and has the peptide synthetic activity; and [3] the protein including the amino acid sequence having 80% or more of the same homology as the amino acid sequence and has the peptide synthetic activity.

Description

TECHNICAL FIELD The present invention relates to a method for producing a peptide using a protein having peptide synthesis activity or a transformant expressing the protein. The protein can synthesize a peptide using a free amino acid as a substrate.

L-amino acid ligase (Lal, EC 6.3.2.28) is a microorganism-derived enzyme capable of synthesizing a peptide by coupling with a hydrolysis reaction of ATP using a free amino acid having no protecting group as a substrate (Non-Patent Document 1). And 2).

By using a recombinant microorganism that expresses the Lal gene, it is possible to synthesize the peptide by a fermentation method (Non-Patent Document 3). Therefore, Lal is an enzyme that is extremely useful industrially. No, there have been reports of several types of Lal so far (Non-patent documents 4 to 7). However, due to the substrate specificity of Lal, it is not possible to efficiently produce all of the 400 dipeptides that are combined from the 20 amino acids that make up the protein.

Tabtoxin is a compound known as a plant pathogenic peptide derived from Pseudomonas syringae , and a Tabtoxin biosynthetic cluster has also been identified in the P. syringe BR2 strain (Non-patent Document 8). However, it was not known what function each gene product has, and it was not known whether the L-amino acid ligase was included in this cluster.

J. Bacteriology, 81, 13-22 (2008) J. Bacteriology, 187, 5195-5202 (2005) Appl. Environ. Microbiol., 73, 6378-6285 (2007) Biosci. Biotechnol. Biochem., 273, 901-907 (2009) Biosci. Biotechnol. Biochem., 72, 3048-3050 (2008) J. Biosci. Bioeng., 106, 313-315 (2008) Biochem. Biophys. Res. Commun., 371, 536-540 (2008) J. Antibiot. 58, 817-821 (2005)

An object of the present invention is to provide a method for producing a peptide using a protein having a peptide-synthesizing activity as an enzyme source, a culture of a transformant expressing a protein having the peptide-synthesizing activity, or a treated product of the culture as an enzyme source. It is intended to provide a method for producing a peptide used and a method for producing a peptide by culturing a transformant expressing the protein.

The present invention relates to the methods described in (1) to (8) below.
(1) The protein according to any of the following [1] to [3], one or more kinds of amino acids, and ATP are allowed to exist in an aqueous medium, and a peptide is produced and accumulated in the medium, A method for producing a peptide, which comprises collecting the peptide.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity (2) ATP is ATP produced by polyphosphate kinase. The method according to (1) above, which is characterized.
(3) A culture of a transformant expressing the protein according to any one of the following [1] to [3] or a treated product of the culture, one or more amino acids, and ATP present in an aqueous medium. And a peptide is produced and accumulated in the medium, and the peptide is collected from the medium.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein (4) transformant comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity is represented by the following [1] to [3 ] The method for producing a peptide according to (3) above, which is transformed with the DNA according to any one of the above.
[1] DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3
[2] A DNA consisting of a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1 or 3 and encoding a protein having peptide synthesis activity
[3] A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encodes a protein having peptide synthesis activity
(5) The method according to (3) or (4) above, wherein ATP is ATP produced by polyphosphate kinase.
(6) A transformant that expresses the protein according to any one of [1] to [3] below and has the ability to produce one or more amino acids is cultured in a medium, and the peptide is added to the culture. A method for producing a peptide, which comprises producing and accumulating and collecting the peptide from the culture.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein (7) transformant comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity is represented by the following [1] to [3] The method for producing a peptide according to (3) above, which is transformed with the DNA according to any one of 1.
[1] DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3
[2] A DNA consisting of a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1 or 3, and encoding a protein having peptide synthesis activity
[3] A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encodes a protein having peptide synthesis activity

According to the present invention, a method for efficiently producing a peptide using a free amino acid as a substrate is provided.

1. Protein used in the production method of the present invention Examples of the protein used in the production method of the present invention include proteins having the amino acid sequences described in any of the following [1] to [3].
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity

In the above, a protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted or added, and having peptide synthesis activity is described in Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (1989). (Hereinafter abbreviated as Molecular Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10 , 6,487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Using the site-directed mutagenesis method described in Sci. USA, 82 , 488 (1985), etc., for example, to introduce a site-directed mutation into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. Can be obtained.

The number of amino acid residues to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a well-known method such as the above site-specific mutation method, and is 1 to several tens. The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

The deletion, substitution or addition of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 2 means 1 to several tens, preferably 1 to 20 at any position in the same sequence. , More preferably 1 to 10 and even more preferably 1 to 5 amino acid residues may be deleted, substituted or added.

Examples of the positions of amino acids at which amino acid residues can be deleted or added include 10 amino acid residues on the N-terminal side and the C-terminal side of the amino acid sequence represented by SEQ ID NO:2.

Deletions, substitutions or additions may occur at the same time, and the substituted or added amino acids may be natural or non-natural. Examples of natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine and L. -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Below, the example of the amino acid which can be mutually substituted is shown. Amino acids included in the same group can be substituted with each other.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid C group: asparagine, glutamine D group: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid E group: proline, 3 -Hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine

Further, as a protein having peptide synthesis activity, the homology with the amino acid sequence represented by SEQ ID NO: 2 is 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly It is preferably a protein consisting of an amino acid sequence having homology of 98% or more, most preferably 99% or more, and a protein having the activity of synthesizing tabtoxin in cooperation with a protein encoded by another tabtoxin biosynthetic cluster. I can give you.

For homology of amino acid sequences and nucleotide sequences, the algorithms BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods Enzymol., 183 , 63 (1990)] by Karlin and Altschul were used. Can be decided. Programs called BLASTN and BLASTX have been developed based on this algorithm BLAST [J. Mol. Biol., 215 , 403 (1990)]. When the nucleotide sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score=100 and wordlength=12. When the amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score=50 and wordlength=3. Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402) to obtain gapped alignment. Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects positional relationships (Id.) between molecules and relationships between molecules that share a common pattern. When using the BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs can be used (see http://www.ncbi.nlm.nih.gov.).

In the present invention, the peptide synthesis activity means an activity of producing a peptide by using adenosine-5'-triphosphate (hereinafter, abbreviated as ATP) using amino acid as a substrate, and preferably L-amino acid, glycine or peptide Refers to the activity of forming a peptide by the reaction of binding the C-terminus of L-amino acid, glycine or the N-terminus of a peptide.

As a means for confirming that the protein used in the production method of the present invention is a protein having peptide synthesis activity, a transformant expressing the protein having peptide synthesis activity is prepared by using, for example, DNA recombination method. After purifying the protein of the present invention using the transformant, the purified protein is used as an enzyme source to allow the enzyme source, one or more amino acids, and ATP to exist in an aqueous medium. A method of analyzing by HPLC or the like whether or not a peptide is produced and accumulated therein can be mentioned.

2. DNA used in the production method of the present invention
The DNA used in the production method of the present invention includes
[4] DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3.
[5] A DNA encoding a protein having 80% or more homology with the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3, and [6] SEQ ID NO: 1 or 3. A DNA that hybridizes with a DNA having a base sequence complementary to the represented base sequence under stringent conditions and that encodes a protein having peptide synthesis activity;
Can be raised.

The term "hybridize" as used herein refers to a step in which the DNA has a specific base sequence or a part of the DNA hybridized with the DNA. Therefore, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or has a length that can be used as an oligonucleotide primer for PCR analysis. May be. The DNA used as a probe for Northern or Southern blot analysis can be at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more, and at least 10 bases can be used as the DNA used as the oligonucleotide primer. As mentioned above, DNA having preferably 15 bases or more can be mentioned.

Methods for DNA hybridization experiments are well known, for example, Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press( In addition to those described in (Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

The stringent conditions are, for example, 50% formamide of a filter on which DNA is immobilized and probe DNA, 5×SSC (750 mM sodium chloride, 75 mmol/L sodium citrate), 50 mmol/L sodium phosphate. (PH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/L denatured salmon sperm DNA after incubation at 42° C. overnight, eg, 0.2× at about 65° C. The conditions for washing the filter in the SSC solution can be raised, but lower stringent conditions can also be used. The stringent condition can be changed by adjusting the concentration of formamide (the lower the concentration of formamide, the lower the stringency), the salt concentration and the temperature condition. Low stringent conditions include, for example, 6×SSCE (20×SSCE is 3 mol/L sodium chloride, 0.2 mol/L sodium dihydrogen phosphate, 0.02 mol/L EDTA, pH 7.4), 0.5% Incubate at 37°C overnight in a solution containing SDS, 30% formamide, and 100 μg/L denatured salmon sperm DNA, then wash with 50°C 1xSSC, 0.1% SDS solution. I can give you. Further, as the lower stringent condition, there may be mentioned the condition of washing after performing hybridization using a solution having a high salt concentration (for example, 5×SSC) under the above-mentioned low stringent condition.

The various conditions described above can be set by adding or changing a blocking reagent used for suppressing the background of the hybridization experiment. The addition of the blocking reagents described above may be accompanied by changes in hybridization conditions in order to adapt the conditions.

Examples of the DNA hybridizable under the above stringent conditions include, for example, the bases of the DNA described in [4] above when calculated based on the above parameters using the programs such as BLAST and FASTA described above. A DNA having at least 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, particularly preferably 99% or more homology with the sequence can be mentioned.

The homology of nucleotide sequences can be determined using a program such as BLAST or FASTA described above.

The fact that the DNA that hybridizes with the above-mentioned DNA under stringent conditions is a DNA encoding a protein having peptide synthetic activity means that a recombinant DNA that expresses the DNA is prepared and The protein is purified from a culture obtained by culturing a transformant obtained by introducing the enzyme into a host cell, and using the purified protein as an enzyme source, the enzyme source and one or more amino acids, amino acid derivatives Alternatively, it can be confirmed by allowing a peptide to exist in an aqueous medium and analyzing by HPLC or the like whether or not the peptide is produced and accumulated in the aqueous medium.

3. The transformant used in the production method of the present invention is not particularly limited as long as it can be used in the production method of the present invention, as long as it can be used in the production method of the present invention, the above [ Using a transformant expressing the protein according to any one of 1] to [3] and a recombinant DNA containing the DNA according to any of the above [4] to [6], a known host cell is used. The transformant obtained by transforming by the method can be mentioned. Examples of host cells include prokaryotes such as bacteria, and preferably microorganisms belonging to the genus Escherichia .

4. Preparation of DNA used in the production method of the present invention The DNA used in the production method of the present invention uses Pseudomonas ( Pseudomonas ) using a probe or primer that can be designed based on the nucleotide sequence represented by SEQ ID NO: 1 or 3. Microorganisms belonging to the genus, preferably Pseudomonas syringe , more preferably Southern hybridization for the chromosome of P. syringe NBRC14081 strain, or a method such as PCR using the chromosome as a template [PCR Protocols, Academic Press (1990)] Can be obtained by

Further, it is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% with the nucleotide sequence of the DNA encoding the amino acid sequence represented by SEQ ID NO: 1 or 3 in various gene sequence databases. % Or more preferably 99% or more homologous sequences, and based on the base sequence obtained by the search, the chromosomal DNA of the organism having the base sequence, cDNA library, etc. It is also possible to obtain the DNA used in the production method of the invention.

The obtained DNA is directly or after cleaved with an appropriate restriction enzyme and the like, and incorporated into a vector by a conventional method, and the obtained recombinant DNA is introduced into a host cell, and then a commonly used nucleotide sequence analysis method, for example, dideoxy method [ Proc. Natl. Acad. Sci., USA, 74 , 5463 (1977)] or a nucleotide sequence analyzer such as ABI3700 DNA Analyzer (manufactured by Applied Biosystems) to determine the nucleotide sequence of the DNA. can do.

When the obtained DNA has a partial length as a result of determining the nucleotide sequence, the full-length DNA can be obtained by the Southern hybridization method or the like for a chromosomal DNA library using the partial length DNA as a probe. ..

Furthermore, the target DNA can also be prepared by chemical synthesis based on the determined nucleotide sequence of the DNA using a Perceptive Biosystems Model 8905 DNA synthesizer or the like.

As the DNA obtained as described above, for example, a DNA having the base sequence represented by SEQ ID NO: 1 can be mentioned.

As a vector incorporating the DNA used in the production method of the present invention, pBluescriptII KS(+) (manufactured by Stratagene), pDIRECT[Nucleic Acids Res., 18 , 6069 (1990)], pCR-Script Amp SK(+). (Stratagene), pT7Blue (Novagen), pCR II (Invitrogen) and pCR-TRAP (Gene Hunter).

Examples of host cells include microorganisms belonging to the genus Escherichia. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli W1485, Escherichia.・Coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415 etc. it can.

As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing the DNA into the above-mentioned host cell. For example, a method using calcium ion [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)], the protoplast method (JP-A-63-248394), the electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.

Examples of the transformant of the present invention obtained by the above method include Escherichia coli BL21 (DE3)/pTABS which is a microorganism having a recombinant DNA containing a DNA having the nucleotide sequence represented by SEQ ID NO:1. I can give you.

5. Method for Producing Transformant Used in Production Method of the Present Invention Based on the DNA used in the present invention, a DNA fragment having an appropriate length containing a protein-encoding portion is prepared, if necessary. Further, by substituting the bases of the nucleotide sequence of the portion encoding the protein so that the codons are optimal for host expression, a transformant with an improved production rate of the protein can be obtained.

A recombinant DNA is prepared by inserting the DNA fragment into the downstream of the promoter of an appropriate expression vector.

A transformant producing the protein of the present invention can be obtained by introducing the recombinant DNA into a host cell suitable for the expression vector.

Any prokaryotic organism such as bacteria can be used as the host cell.
As the expression vector, one that can autonomously replicate in the above host cell or can be integrated into the chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

Recombinant DNA used for obtaining transformants is a set composed of a promoter, a ribosome binding sequence, a DNA encoding a protein having peptide synthesizing activity, and a transcription termination sequence while being capable of autonomous replication in a prokaryote. It is preferably a recombinant DNA. A gene that controls the promoter may be included.

As the expression vector, pBTrp2, pBTac1, pBTac2, pHelix1 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech), pSE280 (manufactured by Invitrogen), pGEMEX-1 (promega) Manufactured), pQE-8 (manufactured by Qiagen), pET-3 (manufactured by Novagen), pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 48 , 669 (1984)], pLSA1[ Agric. Biol. Chem., 53 , 277 (1989)], pGEL1[Proc. Natl. Acad. Sci., USA, 82 , 4306 (1985)], pBluescriptII SK(+), pBluescript II KS(-) (Strata) Gene), pTrS30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pPAC31 (WO98/12343), pUC19 [ Gene, 33 , 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), pPA1 (JP-A-63-233798) and the like.

Any promoter may be used so long as it can function in a host cell such as Escherichia coli. Examples include promoters derived from Escherichia coli and phages such as trp promoter (P _trp ), lac promoter (P _lac ), P _L promoter, P _R promoter and P _SE promoter, SPO1 promoter, SPO2 promoter, penP promoter and the like. be able to. Further, artificially designed and modified promoters such as a promoter in which two P _trp are connected in series, tac promoter, lacT7 promoter, let I promoter and the like can also be used.

The xylA promoter [Appl. Microbiol. Biotechnol., 35 , 594-599 (1991)] for expression in microorganisms belonging to the genus Bacillus and P54- for expression in microorganisms belonging to the genus Corynebacterium 6 promoter [Appl. Microbiol. Biotechnol., 53 , 674-679 (2000)] and the like can also be used.

In addition, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

In the recombinant DNA in which the DNA of the present invention is bound to the expression vector, the transcription termination sequence is not always necessary, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

The prokaryotic organisms, Escherichia, Bacillus, Brevibacterium (Brevibacterium), Corynebacterium, micro Agrobacterium (Microbacterium) genus Serratia (Serratia) genus Pseudomonas (Pseudomonas) genus, Agrobacterium (Agrobacterium) genus Alicyclobacillus Bacillus (Alicyclobacillus) genus Anabaena (Anabena) genus Anashisutisu (Anacystis) genus Arthrobacter (Arthrobacter) genus Azotobacter (Azotobacter) genus Kuromachiumu (Chromatium) genus Erwinia (Erwinia) genus, methylol bacterium (Methylobacterium) genus, Phormidium (Phormidium) genus Rhodobacter (Rhodobacter) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Rodosupiriumu (Rhodospirillum) genus Scenedesmus (Scenedesmus) genus Streptomyces (Streptomyces) genus, Shinekokkasu ( Synechoccus) genus Zymomonas (Zymomonas) microorganism belonging to the genus, such as, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli BL21 (Bacillus subtilis) ATCC33712, Bacillus megaterium (Bacillus megaterium), Brevibacterium ammonia monocytogenes (Brevibacterium ammoniagenes), Brevibacterium Lee Mario film (Brevibacterium immariophilum) ATCC14068, Burebibakute Potassium Sacca Lori tee cam (Brevibacterium saccharolyticum) ATCC14066, Brevibacterium flavum (Brevibacterium flavum) ATCC14067, Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869, Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, Corynebacterium glutamicum ATCC14297, Corynebacterium acetamide A Sid film (Corynebacterium acetoacidophilum) ATCC13870, micro Corynebacterium ammoniagenes film (Microbacterium ammoniaphilum) ATCC15354, Serratia Fikaria (Serratia ficaria), Serratia Fonchikora (Serratia fonticola), Serratia Rikefashiensu (Serratia liquefaciens), Serratia marcescens (Serratia marcescens), Pseudomonas sp. (Pseudomonas sp.) D-0110 , Agrobacterium radiobacter (Agrobacterium radiobacter), Agrobacterium Rizzo jeans (Agrobacterium rhizogenes), Agrobacterium ruby (Agrobacterium rubi), Anabaena-Shirindorika (Anabaena cylindrica), Anabaena-Doriorumu (Anabaena doliolum), Anabaena floss Aqua (Anabaena flos-aquae), Arthrobacter Ore' sense (Arthrobacter aurescens), Arthrobacter citreus (Arthrobacter citreus ), Arthrobacter globformis , Arthrobacter hydrocarboglutamicus , Arthrobac ter mysorens), Arthrobacter Nicotiana (Arthrobacter nicotianae), Arthrobacter paraffineus (Arthrobacter paraffineus), Arthrobacter proto folder Mie (Arthrobacter protophormiae), Arthrobacter Rose opal Rafi eggplant (Arthrobacter roseoparaffinus), ground Robakuta sulphureus (Arthrobacter sulfureus), Arthrobacter urea tumefaciens (Arthrobacter ureafaciens), Kuromachiumu-Buderi (Chromatium buderi), Kuromachiumu-Tepidamu (Chromatium tepidum), Kuromachiumu-Binosamu (Chromatium vinosum), Kuromachiumu-Wamingi (Chromatium warmingii), Kuromachiumu-Furubiatatire (Chromatium fluviatile), Erwinia Uredobara (Erwinia uredovora), Erwinia Karotobara (Erwinia carotovora), Erwinia Anas (Erwinia ananas), Erwinia Herikora (Erwinia herbicola), Erwinia Pankutata (Erwinia punctata) , Erwinia terreus (Erwinia terreus), Methylobacterium-Rodeshianamu (Methylobacterium rhodesianum), Methylobacterium-exo torque Enns (Methylobacterium extorquens), Phormidium sp (Phormidium sp.) ATCC29409, Rhodobacter Kapusuratasu (Rhodobacter capsulatus), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rod Pseudomonas Burasuchika (Rhodopseudomonas blastica), Rod Pseudomonas Marina (Rhodopseudomonas marina), Rod Pseudomonas pulse tris (Rhodopseudomonas palustris), Rodosupiriumu-Riburamu (Rhodospirillum rubrum), Rodosupiriumu-Sarekishigensu (Rhodospirillum salexigens), Rodosupiriumu-Sarinaramu (Rhodospirillum salinarum), Streptomyces ambofaciens (Streptomyces ambofaciens), Streptomyces aureofaciens (Streptomyces aureofaciens ), Streptomyces aureus , Streptomyces fungicidicus , Streptomyces griseochromogenes , Streptomyces griseus , Streptomyces griseus (Streptomyces lividans, Streptomyces Oriboguriseusu (Streptomyces olivogriseus), Streptomyces Rameusu (Streptomyces rameus), Streptomyces Tanashienshisu (Streptomyces tanashiensis), Streptomyces Binaseusu (Streptomyces vinaceus), Zymomonas mobilis (Zymomonas mobilis ) And so on.

As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing the DNA into the above-mentioned host cell. For example, a method using calcium ion [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)], the protoplast method (JP-A-63-248394), the electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.

A transformant expressing a protein having peptide synthesis activity can also be obtained by incorporating a DNA encoding a protein having peptide synthesis activity at an arbitrary position of chromosomal DNA.

As a method for incorporating a DNA encoding a protein having peptide synthesis activity into a chromosomal DNA of a microorganism, a method utilizing homologous recombination can be mentioned. When E. coli is used as a host, that is, a parent strain. Can be exemplified by the method described in Proc. Natl. Acad. Sci. US A., 97 , 6640 (2000).

6. Method for producing peptide of the present invention (1) Acquisition of protein having peptide synthesis activity The protein having the peptide synthesis activity of the above-mentioned 5. It can be obtained by culturing the transformant obtained by the above method in a medium and isolating the protein from the culture.
Cultivation of the transformant is carried out using a natural medium or a synthetic medium culture containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the transformant and capable of efficiently culturing the transformant. be able to.

The carbon source may be any as long as the transformant can assimilate, glucose, fructose, sucrose, xylose, arabinose molasses containing these, saccharified liquid of cellulosic biomass, glycerol, starch or starch hydrolyzate, etc. The organic acids such as carbohydrates, acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.

As a nitrogen source, ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, casein Hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells, digested products thereof, and the like can be used.

As the inorganic salt, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40° C., and the culturing time is usually 5 hours to 7 days. The pH is maintained at 3.0 to 9.0 during the culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

In addition, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culturing, if necessary.

When culturing a transformant having an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a transformant having an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used, and when culturing a transformant having an expression vector using the trp promoter, indole acrylic is used. You may add an acid etc. to a culture medium.

Above 1. The protein can be produced in the host cell, secreted to the outside of the host cell, or produced on the outer membrane of the host cell, depending on the method selected. Can be changed.

Above 1. When the protein of E. coli is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. [J. Biol. Chem., 264 , 17619 (1989)], the method of Law et al. [Proc. Natl. Acad. Sci ., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], or the method described in JP-A-05-336963, WO94/23021, etc. Can be secreted outside the host cell.

That is, by using a gene recombination technique, a protein containing the active site of the protein is produced in the form in which a signal peptide is added in front of the protein, whereby the protein can be actively secreted outside the host cell.

Further, according to the method described in JP-A-2-227075, the production amount can be increased by utilizing a gene amplification system using a dihydrofolate reductase gene or the like.

The above 1. produced by using the transformant. As a method for isolating and purifying the protein having the peptide-synthesizing activity, the usual enzyme isolation and purification methods can be used.

For example, when the protein is produced in a dissolved state in cells, the cells are recovered by centrifugation after the completion of culturing, suspended in an aqueous buffer solution, ultrasonically disrupted, French press, Mantongaurin homogenizer. The cells are crushed with, for example, Dynomill to obtain a cell-free extract.

From the supernatant obtained by centrifuging the cell-free extract, a usual method for isolating and purifying an enzyme, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino, etc. Anion exchange chromatography using resins such as ethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), cations using resins such as S-Sepharose FF (manufactured by GE Healthcare Bioscience) Exchange chromatography method, hydrophobic chromatography method using resins such as butyl sepharose and phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoretic method such as isoelectric focusing, etc. A purified standard can be obtained by using the above methods alone or in combination.

In addition, the above 1. When the protein of (1) is produced by forming an insoluble substance in the cells, cells are similarly collected, crushed, and then centrifuged to perform precipitation, and then the protein is recovered by an ordinary method. , The insoluble matter of the protein is solubilized with a protein denaturing agent.

After diluting or dialysis the solubilized solution into a solution that does not contain a protein denaturant or a protein denaturant is diluted to such an extent that the protein does not denature, or the protein is made into a normal three-dimensional structure, A purified preparation can be obtained by the same isolation and purification method.

Above 1. When the above protein is secreted extracellularly, the protein or its derivative such as a glycosylated product can be recovered in the culture supernatant.

That is, a soluble preparation is obtained by treating the culture with a method such as centrifugation similar to the above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.

Examples of the protein thus obtained include a protein having the amino acid sequence represented by SEQ ID NO:2.

It is also possible to produce the above protein 1 as a fusion protein with another protein and purify it using affinity chromatography using a substance having an affinity for the fused protein. For example, the method described in Low et al. [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], JP-A-5-336963, WO94/23021. In accordance with 1. above. Can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

In addition, the above 1. Affinity chromatography using the anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990). Alternatively, it can be produced as a fusion protein with polyhistidine and purified by affinity chromatography using a metal coordination resin having a high affinity for polyhistidine. Furthermore, it can be purified by affinity chromatography using an antibody against the protein itself.

The protein of the present invention is produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) based on the amino acid sequence information of the protein obtained above. be able to. In addition, chemical synthesis can also be performed using a peptide synthesizer such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu.

(2) The above 1. Method for producing a peptide using the above protein as an enzyme source In the production method of the present invention, when the above protein 1 is used as an enzyme source, one or more amino acids used as a substrate, preferably 1 or 2 amino acids, more preferably Is an amino acid selected from the group consisting of amino acids, preferably L-amino acids, glycine (Gly) and β-alanine (β-Ala), any amino acid may be used in any combination. Good. Examples of L-amino acids include L-alanine (L-Ala), L-glutamine (L-Gln), L-glutamic acid (L-Glu), L-valine (L-Val) and L-leucine (L-Leu). ), L-isoleucine (L-Ile), L-proline (L-Pro), L-phenylalanine (L-Phe), L-tryptophan (L-Trp), L-methionine (L-Met), L-serine (L-Ser), L-Threonine (L-Thr), L-Cysteine (L-Cys), L-Asparagine (L-Asn), L-Tyrosine (L-Tyr), L-Lysine (L-Lys) , L-arginine (L-Arg), L-histidine (L-His), L-aspartic acid (L-Asp), L-homoarginine (L-HomoArg), L-α-aminobutyric acid, L-azaserine, Examples thereof include L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline and L-6-diazo-5-oxo-norleucine.

As a preferred amino acid used in the above-mentioned production method, L-Ala, L-Gln, L-Glu, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L- Mention may be made of Met, L-Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala.

More preferred amino acids include a combination of L-Cys and L-Met or L-Leu, L-Met and L-Ala, L-Gln, L-Glu, L-Val, L-Leu, L-Ile, L. -Pro, L-Phe, L-Trp, L-Met, L-Ser, L-Thr, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, Gly and β -Combination with an amino acid selected from Ala, L-Val and L-Ala, L-Gln, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Ser, A combination with an amino acid selected from L-Thr, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, Gly and β-Ala, L-Leu and L-Ala, L-Gln, L -Leu, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Asn, L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala L-Ile and L-Ala, L-Gln, L-Ile, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Asn, L-Tyr, Combination with an amino acid selected from L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala, L-Ala and L-Ala, L-Gln, L-Pro, L-Phe, L -A combination with an amino acid selected from Trp, L-Ser, L-Thr, L-Tyr, L-Arg, L-His, L-Asp and β-Ala, Gly and L-Pro, L-Phe or L- In combination with Trp, L-Ser with an amino acid selected from L-Gln, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Asn, L-Arg and L-His From combinations, L-Thr and L-Gln, L-Pro, L-Phe, L-Trp, L-Thr, L-Asn, L-Tyr, L-Lys, L-Arg, L-His and β-Ala Combination with selected amino acids, L-Arg and L-Gln, L-Pro, L-Phe, L-Trp, L-Asn, L-Tyr, L-Lys, L-Arg, L-His and β-Ala A combination with an amino acid selected from, a combination of L-Lys and L-Trp or L-Phe, a combination with L-His and an amino acid selected from L-Gln, L-Phe, L-Trp and L-Tyr, L -Gln and L-Pro, L-Phe, L-Trp, A combination with an amino acid selected from L-Asn and L-Tyr, a combination with L-Asn and L-Pro, a combination with L-Phe or L-Trp, a combination with L-Asp and L-Trp, L-Pro and L -A combination of amino acids selected from Pro, L-Phe, L-Trp and β-Ala, a combination of L-Trp and L-Pro, L-Phe, L-Trp or β-Ala, L-Phe and L Only -Phe or L-Tyr in combination, as well as β-Ala can be mentioned.

More preferred amino acids are L-Ala and L-Pro combination, L-Leu and L-Pro combination, L-Val and L-Pro combination, L-Pro only, and L-Phe and β-Ala combination. Can be raised.

In the above production method, the protein having peptide synthesis activity is added in an amount of 0.01 to 100 mg, preferably 0.1 mg to 10 mg, per 1 mg of the amino acid used as the substrate.

In the above-mentioned production method, the amino acid used as a substrate is added to the aqueous medium at the initial stage or during the reaction so that the concentration thereof is 0.1 to 500 g/L, preferably 0.2 to 200 g/L.

In the above production method, ATP can be used as an energy source, and ATP is used at a concentration of 0.5 mmol to 10 mol/L.

The aqueous medium used in the above production method may be an aqueous medium of any component or composition as long as it does not inhibit the peptide production reaction, and examples thereof include water, phosphate, carbonate, acetate and borate. Buffers such as citrate and Tris. Further, it may contain alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide.

The peptide-forming reaction is carried out in an aqueous medium under the conditions of pH 5 to 11, preferably pH 6 to 10, 20 to 50° C., preferably 25 to 45° C. for 2 to 150 hours, preferably 6 to 120 hours.

The peptide produced by the above method, L-Ala, L-Gln, L-Glu, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Met, Amino acids selected from L-Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala are linked by peptide bonds Dipeptides, preferably L-Cys and L-Met, L-Met and L-Ala, L-Gln, L-Glu, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L -Amino acid selected from Trp, L-Met, L-Ser, L-Thr, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala, L -Val and L-Ala, L-Gln, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Asn, L-Tyr , L-Lys, L-Arg, L-His, Gly and β-Ala, L-Leu and L-Ala, L-Gln, L-Leu, L-Pro, L-Phe, L-Trp , L-Ser, L-Thr, L-Asn, L-Lys, L-Arg, L-His, L-Asp, Gly and β-Ala, L-Ile and L-Ala, L-Gln , L-Ile, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp, Gly And amino acids selected from β-Ala, L-Ala and L-Ala, L-Gln, L-Pro, L-Phe, L-Trp, L-Ser, L-Thr, L-Tyr, L-Arg, L -An amino acid selected from His, L-Asp and β-Ala, Gly and L-Pro, L-Phe or L-Trp, L-Ser and L-Gln, L-Pro, L-Phe, L-Trp, L -Amino acids selected from Ser, L-Thr, L-Asn, L-Arg and L-His, L-Thr and L-Gln, L-Pro, L-Phe, L-Trp, L-Thr, L-Asn , L-Tyr, L-Lys, L-Arg, L-His and β-Ala, L-Arg and L-Gln, L-Pro, L-Phe, L-Trp, L-Asn, L -From Tyr, L-Lys, L-Arg, L-His and β-Ala Amino acids selected, L-Lys and L-Trp or L-Phe, L-His and L-Gln, L-Phe, L-Trp and L-Tyr selected amino acids, L-Gln and L-Pro, L- Phe, an amino acid selected from L-Trp, L-Asn and L-Tyr, L-Asn and L-Pro, L-Phe or L-Trp, L-Asp and L-Trp, L-Pro and L-Pro, Amino acids selected from L-Phe, L-Trp and β-Ala, L-Trp and L-Pro, L-Phe, L-Trp or β-Ala, L-Phe and L-Phe or L-Tyr, and β -A dipeptide in which Ala is linked by a peptide bond, a tripeptide in which three L-Leus are linked by a peptide bond, a tripeptide in which three L-Mets are linked by a peptide bond, two L-Leu and L-Met, L -A tripeptide in which amino acids selected from Arg, L-Ala, L-Lys, L-His, L-Pro and L-Phe are linked by a peptide bond, and two L-Phe and L-Leu are linked by a peptide bond A tripeptide can be mentioned.

More preferably, L-Ala and L-Pro, L-Leu and L-Pro, L-Val and L-Pro, L-Pro only, and a dipeptide in which L-Phe and β-Ala are linked by a peptide bond. be able to.

More specifically, as the peptide produced by the above method, L-Pro-L-Ala, L-Pro-L-Leu, L-Pro-L-Val, L-Pro-L-Pro, L- Ala-L-Ala, L-Leu-L-Leu and β-Ala-L-Phe can be mentioned.

(3) Method for producing peptide using culture of transformant or treated product of culture as an enzyme source Of a transformant expressing the above protein or a treated product of the culture, at least one amino acid, and ATP are allowed to exist in an aqueous medium, and a peptide is produced and accumulated in the medium, and the peptide is produced from the medium. Can be isolated to produce the peptide.

Examples of the culture of the transformant used as the enzyme source in the production method of the present invention include the culture obtained by culturing the transformant by the culture method of (1) above. As a processed product of the transformant culture, a concentrate of the culture, a dried product of the culture, cells obtained by centrifuging or filtering the culture, a dried product of the cells, The lyophilized product of the microbial cells, the surfactant-treated product of the microbial cells, the solvent-treated product of the microbial cells, the enzyme-treated product of the microbial cells, and the culture as an enzyme source of an immobilized product of the microbial cells Those containing live cells having the same function, sonicated products of the microbial cells, mechanically milled products of the microbial cells, and crude enzyme extracts obtained from the treated microbial cells, etc. Can be raised.

When a culture product of a transformant or a treated product of the culture product is used as an enzyme source, the same amino acid as in (2) above can be used as the one or more amino acids used as the substrate.

Although the amount of the enzyme source varies depending on the specific activity of the enzyme source and the like, for example, 5 to 1000 mg, preferably 10 to 400 mg as the weight of wet cells per 1 mg of the amino acid used as a substrate is added.

The amino acid used as the substrate can be added to the aqueous medium in the same manner as (2) above. Similar to (2) above, ATP can be allowed to exist in an aqueous medium and used as an energy source.

ATP can also be supplied by allowing polyphosphate kinase to coexist in the above aqueous medium. The polyphosphate kinase activity may be derived from any protein having such activity, regardless of its origin, preparation method and the like. For example, Agric. Biol. Chem., 52 (6), 1471-1477 (1988), Biotech. Appl. Biochem ., 10 , 107-117 (1988), Biotech. Appl. Biochem., 15 , 125-133 (1992), J. Biol. Chem., 267 , 22556-22561 (1992), etc. You can mention acid kinase.

ATP regenerates ATP from ADP by allowing the above-mentioned polyphosphate kinase, polyphosphate, and a small amount of AMP, ADP or ATP to the extent that the reaction of polyphosphate kinase starts at the beginning of the reaction, in an aqueous medium. The reaction proceeds and ATP can be continuously supplied to the reaction system.

As the polyphosphate kinase, a culture of a microorganism producing polyphosphate kinase or a treated product of the culture itself can be used as an enzyme source. The method for culturing the microorganism and the treated product of the culture are the same as above.

As the aqueous medium, the medium of (2) above can be used, and in addition, the culture supernatant of the culture of the microorganism or transformant used as the enzyme source can also be used as the aqueous medium.

The reaction conditions for the peptide production reaction can be the same as those in (2) above.
Examples of the peptide produced by the above method include the same peptides as in (2) above.

(4) Method for producing peptide by culturing transformant The above 1. By culturing a transformant having the ability to express the above protein and capable of producing one or more amino acids in a medium, to produce and accumulate the peptide in the culture, and to collect the peptide from the culture. Peptides can be produced.

Cultivation of the transformant can be performed in the same manner as in (1) above.
A transformant having the ability to produce one or more amino acids can be obtained by directly transforming the transformant if the host cell that is the source of the transformant already has the ability to produce one or more amino acids. The peptide can be produced by culturing.

When the host cell does not have the ability to produce an amino acid, the host cell is artificially endowed with the ability to produce one or more amino acids, or the above 1. The transformant obtained by transforming with the DNA encoding the protein of 1. above is used in the above production method by imparting the ability to produce one or more amino acids. It is possible to obtain a transformant having the ability to express the above protein and produce one or more amino acids.

The one or more amino acids include L-Ala, L-Gln, L-Glu, L-Val, L-Leu, L-Ile, L-Pro, L-Phe, L-Trp, L-Met, L-. One or more amino acids selected from Ser, L-Thr, L-Cys, L-Asn, L-Tyr, L-Lys, L-Arg, L-His, L-Asp and Gly can be mentioned.

As a method of artificially imparting the ability to produce amino acids,
(A) a method of relaxing or releasing at least one of the mechanisms controlling amino acid biosynthesis,
(B) a method of enhancing expression of at least one of enzymes involved in biosynthesis of amino acids,
(C) a method of increasing the copy number of at least one enzyme gene involved in amino acid biosynthesis,
(D) a method of weakening or blocking at least one of metabolic pathways branching from an amino acid biosynthetic pathway to a metabolite other than the amino acid, and (e) a cell having a high degree of resistance to an amino acid analog as compared to a wild-type strain How to choose a stock,
The above known methods can be used alone or in combination.

Regarding the above (a), for example, Agric. Biol. Chem., 43 , 105-111 (1979), J. Bacteriol., 110 , 761-763 (1972) and Appl. Microbiol. Biotechnol., 39 , 318-323. (1993) and the like, for (b) above, see, for example, Agric. Biol. Chem., 43 , 105-111 (1979) and J. Bacteriol., 110 , 761-763 (1972), above (c). For example, Appl. Microbiol. Biotechnol., 39 , 318-323 (1993) and Agric. Biol. Chem., 39 , 371-377 (1987), and the above (d), for example, Appl. Environ. Micribiol., 38 , 181-190 (1979) and Agric. Biol. Chem., 42 , 1773-1778 (1978) and the like, for (e) above, for example, Agric. Biol. Chem., 36 , 1675-1684. (1972), Agric. Biol. Chem., 41 , 109-116 (1977), Agric. Biol. Chem., 37 , 2013-2023 (1973) and Agric. Biol. Chem., 51 , 2089-2094 (1987). ) Etc. Microorganisms capable of producing various amino acids can be prepared with reference to the above-mentioned documents and the like.

Furthermore, for the method for preparing a microorganism having the ability to produce an amino acid by any of the above (a) to (e), or a combination thereof, Biotechnology 2nd ed., Vol. 6, Products of Primary Metabolism (VCH Verlagsgesellschaft mbH, Weinheim, 1996) section 14a, 14b and Advances in Biochemical Engineering/ Biotechnology 79, 1-35 (2003), Amino Acid Fermentation, Academic Publishing Center, Aida Hiro et al. (1986) have many examples. Also, a method for preparing a microorganism having the ability to produce a specific amino acid is disclosed in JP 2003-164297, Agric. Biol. Chem., 39 , 153-160 (1975), Agric. Biol. Chem., 39 , 1149. -1153 (1975), JP-A-58-13599, J. Gen. Appl. Microbiol., 4 , 272-283 (1958), JP-A-63-94985, Agric. Biol. Chem., 37 , 2013-2023 (1973), WO97/15673, JP-A-56-18596, JP-A-56-144092, and JP-A-2003-511086, and there are many reports, and one or more kinds of amino acids are produced by referring to the above-mentioned documents. Microorganisms capable of being prepared can be prepared.

Examples of microorganisms capable of producing amino acids that can be prepared by the above method include L-alanine-producing microorganisms, microorganisms in which expression of the alanine dehydrogenase gene ( ald gene) is enhanced, and L-proline-producing microorganisms. , Microorganisms that express the desensitized pheA gene of phenylalanine and/or the desensitized aroF gene of tyrosine.

The microorganism that produces and accumulates the above-mentioned amino acid may be any microorganism to which the methods (a) to (e) can be applied or any microorganism having the above-mentioned genetic trait, and it is preferable. Can be prokaryotes, more preferably bacteria.

As prokaryotes, Escherichia (Escherichia) genus Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus Pseudomonas (Pseudomonas) genus, a microorganism belonging to the Streptomyces (Streptomyces) species, such as, for example, Escherichia coli Brevibacterium ammoniagenes , Brevibacterium immariophilum , Brevibacterium saccharolyticum , Brevibacterium flavum , Brevibacterium - Lactobacillus fermentum (Brevibacterium lactofermentum), Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium aceto A Sid film (Corynebacterium acetoacidophilum), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas putida (Pseudomonas putida), Streptomyces aureofaciens (Streptomyces aureofaciens), Streptomyces aureus (Streptomyces aureus), Streptomyces Funjishidikasu (Streptomyces fungicidicus), Streptomyces Gris Theo black Moge eggplant (Streptomyces griseochromogenes), Streptomyces griseus (Streptomyces griseus), Streptomyces lividans (Streptomyces lividans), Streptomyces Oriboguriseusu (Streptomyces olivogriseus), Streptomyces Rameusu (Streptomyces rameus), Streptomyces Tanashienshisu (Streptomyces tanashiensis), strike Examples thereof include Streptomyces vinaceus , and preferred bacteria include Escherichia coli, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium lactofermentum, Corynebacterium flavum, and coryne. Examples thereof include Bacterium efficiens, Pseudomonas putida, Pseudomonas aeruginosa, Streptomyces sericolor or Streptomyces lividans, and particularly preferred is Escherichia coli.

Specific examples of the microorganism that produces an amino acid include Escherichia coli JM101 strain that retains an ald gene expression plasmid as an L-alanine producing strain, and a desensitized pheA gene and/or aroF gene expression plasmid as an L-phenylalanine producing strain. The Escherichia coli JM101 strain, etc., which owns

ATCC13005 and ATCC19561 as L-valine producing strains, FERM BP-4704 and ATCC21302 as L-leucine producing strains, FERM BP-4121 and ATCC15108 as L-alanine producing strains, FERM BP-2807 as L-proline producing strains, and the like. ATCC19224 etc. can be mentioned. The strains represented by the FERM number above should be obtained from the National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center (Japan), and the strains represented by the ATCC number should be obtained from the American Type Culture Collection (USA). You can

In the above production methods (2) to (4), the peptides produced and accumulated in the aqueous medium can be collected by a usual method using activated carbon, an ion exchange resin or the like, or extraction with an organic solvent, crystallization, thin layer chromatography. It can be performed by chromatography, high performance liquid chromatography, or the like.

Examples will be shown below, but the present invention is not limited to the following examples.
In the following examples, analysis of peptides and amino acids by HPLC, quantification, and analysis by LC-ESI-MS were performed by the methods described below.

(1) HPLC analysis Peptides and amino acids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide sodium (Nα-FDAA) and then analyzed by HPLC. Derivatization with Nα-FDAA was carried out by adding 50 μl of 0.5% Nα-FDAA acetone solution and 40 μl of 0.5 mol/l sodium carbonate aqueous solution to 100 μl of a sample diluted with pure water, and stirring the mixture at 40° C. It was performed by leaving it to stand for 60 minutes.

Next, 40 μl of 1 mol/l hydrochloric acid and 770 μl of methanol were added and stirred to obtain an FDAA sample.

The FDAA-converted sample was analyzed and quantified using a WH-C18A column (Hitachi Science Systems, 4×150 mm). The following conditions were used for the conditions of HPLC analysis.

The mobile phase was 50 mmol/l potassium phosphate buffer (pH 2.7, pH adjusted with phosphoric acid), mobile phase A with a mixing ratio of acetonitrile and methanol of 90:5:5, and 50 mmol/l phosphorus. A mobile phase B having a potassium acid buffer solution (pH 2.7, pH adjusted with phosphoric acid) and acetonitrile and methanol at a mixing ratio of 60:35:5, and a mixing ratio of acetonitrile, tetrahydrofuran and water at a ratio of 3:1:3. Mobile phase C was used. The mobile phase flow rate is 0.5 ml/min, the mixing ratio of mobile phase A to mobile phase B and mobile phase C is 0 to 24 minutes, the gradient from 100:0:0 to 55:45:0, 24 to 30 minutes. Is a constant 55:45:0, 30-50 minutes is a slope from 55:45:0 to 0:100:0, 50-55 minutes is a constant 0:100:0, and 55-60 minutes is 0: Slope from 100:0 to 0:0:100, constant 0:0:100 from 60 to 62 minutes, 62 to 62.1 minutes from 0:0:100 to 100:0:0, 62.1 to 80 minutes Changed to a constant of 100:0:0. The column temperature was 40° C., and the ultraviolet absorption at 340 nm was measured.

(2) LC-ESI-MS analysis
A sample was analyzed by a system using a SunFire C18 column (Waters, 4.6×150 mm) as an HPLC separation column and combining LCQ Deca (Thermo Scientific) as ESI-MS.

For the mobile phase, a 50 mmol/l formic acid solution (mobile phase A) and acetonitrile (mobile phase B) were used. The mobile phase flow rate was 1.0 ml/min, the mixing ratio of mobile phase A and mobile phase B was constant at 100:0 for 0 to 2 minutes, and the gradient from 100:0 to 0:100 for 2 to 9 minutes. The gradient was 0:100 for 9 to 15 minutes, the gradient from 0:100 to 100:0 for 15 to 15.1 minutes, and the constant was 100:0 for 15.1 to 20 minutes.

[Example 1]
Cloning of DNA encoding a protein having peptide synthesizing activity and preparation of transformant expressing the protein Pseudomonas syringe NBRC 14801 strain was obtained from Institute for Product Evaluation Technology, Institute of Biological Genetics (NRBC), The chromosomal DNA was extracted by a known method.

Using the DNA having the nucleotide sequences represented by SEQ ID NOS: 4 and 5 as a primer set, PCR was performed using the above chromosomal DNA as a template to obtain the DNA having the nucleotide sequence represented by SEQ ID NO: 1.

The DNA and pET28a(+) were digested with Nde I and Eco RI and then ligated with DNA ligase. The resulting ligated DNA was used to transform Escherichia coli BL21 (DE3) to obtain a transformant strain having a recombinant DNA containing a DNA encoding a protein having peptide synthesis activity. The recombinant DNA was named pTABS and the transformant was named E. coli BL21(DE3)/pTABS.

[Example 2]
Acquisition of protein with peptide synthesis activity
After incubating E. coli BL21(DE3)/pTABS in LB liquid medium containing 25 μg/ml kanamycin at 37°C and 120 rpm for 1-2 hours, add IPTG to a final concentration of 0.1 mmol/l. Then, the cells were cultured at 25° C. and 120 rpm for 16 hours.

The culture was centrifuged to collect the cells, and the cells were suspended in a 100 mmol/l Tris-HCl buffer solution, and then the cells were ultrasonically disrupted. The disrupted cell suspension was centrifuged to remove the cell residue, and the supernatant was collected as a cell-free extract. The extract was passed through a Ni-affinity column (manufactured by GE Healthcare) and purified, and then desalted using a PD-10 column (manufactured by GE Healthcare) to obtain a purified protein. This purified protein was named TabS.

[Example 3]
Production of peptides (1) Production of L-Pro-L-Ala
Prepare a reaction solution consisting of L-Pro 100 mmol/l, L-Ala 20 mmol/l, ATP 50 mmol/l, MgSO ₄ 25 mmol/l, TabS 0.5 mg/ml, Tris HCl 100 mmol/l (pH 9) and 30°C. The peptide synthesis reaction was carried out for 20 hours. The reaction solution supernatant is analyzed under the above-mentioned HPLC conditions, L-Pro-L-Ala 6.3 mmol/l, L-Pro-L-Pro 0.4 mmol/l, L-Ala-L-Ala 0.6 mmol/l. Confirmed generation.

(2) Production of L-Pro-L-Leu
A peptide synthesis reaction was performed under the same conditions as in (1) above, except that L-Ala was changed to L-Leu, and the reaction solution was analyzed by the same method. As a result, production of L-Pro-L-Leu 9.4 mmol/l and L-Leu-L-Leu 1.0 mmol/l was confirmed.

(3) Production of L-Phe-β-Ala
Prepare a reaction solution consisting of L-Phe 40 mmol/l, β-Ala 40 mmol/l, ATP 50 mmol/l, MgSO ₄ 25 mmol/l, TabS 0.5 mg/ml, Tris HCl 100 mmol/l (pH 9) and 30°C. The peptide synthesis reaction was carried out for 20 hours. The reaction solution supernatant was analyzed under the above-mentioned HPLC conditions to confirm the production of L-Phe-β-Ala 0.12 mmol/l.

(4) Production of various peptides From amino acid 1 12.5 mmol/l, amino acid 2 12.5 mmol/l, ATP 12.5 mmol/l, MgSO ₄ 12.5 mmol/l, TabS 0.1 mg/ml, Tris HCl 100 mmol/l (pH 8.0) Was prepared and the peptide synthesis reaction was carried out at 30° C. for 20 hours. Amino acids 1 and 2 are each amino acid shown as amino acid 1 and amino acid 2 in Table 1, respectively. After completion of the reaction, each reaction supernatant was analyzed by LC-ESI-MS described above. The results are shown in Table 1.

When the peptide synthesis reaction was carried out with the combination of amino acids shown in gray in the table, a peak corresponding to the dipeptide was detected in the above system.

(5) Production of tripeptide containing L-Leu and L-Met In the above (4), L-Leu as amino acid 1 and L-Met, L-Arg, L-Ala, L-Lys, L as amino acid 2 -When using His, L-Pro or L-Phe, L-Leu-L-Leu-L-Leu, L-Met-L-Met-L-Met and two L-Leu and L-Met, Two L-Leu and L-Arg, two L-Leu and L-Ala, two L-Leu and L-Lys, two L-Leu and L-His, two L-Leu and L-Pro, Peaks corresponding to tripeptides consisting of two L-Leu and L-Phe and two L-Phe and L-Leu were detected.

By using the present invention, it has become possible to efficiently produce a peptide.

Claims (7)

The protein according to any of the following [1] to [3], one or more amino acids, and ATP are allowed to exist in an aqueous medium, and a peptide is produced and accumulated in the medium, and the peptide is produced from the medium. A method for producing a peptide, which comprises collecting the peptide.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity The method according to claim 1, wherein ATP is ATP supplied by polyphosphate kinase. A culture of a transformant expressing the protein according to any one of the following [1] to [3] or a treated product of the culture, one or more amino acids, and ATP are allowed to exist in an aqueous medium, A method for producing a peptide, which comprises producing and accumulating a peptide in a medium, and collecting the peptide from the medium.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2
[2] A protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, or added, and having peptide synthesis activity
[3] A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity The method for producing a peptide according to claim 3, wherein the transformant has been transformed with the DNA according to any of the following [1] to [3].
[1] DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3
[2] A DNA consisting of a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1 or 3 and encoding a protein having peptide synthesis activity
[3] A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encodes a protein having peptide synthesis activity ATP is ATP produced by polyphosphate kinase, The manufacturing method of Claim 3 or 4 characterized by the above-mentioned. A transformant that expresses the protein according to any of the following [1] to [3] and has the ability to produce one or more amino acids is cultured in a medium, and a peptide is produced and accumulated in the culture. And then collecting the peptide from the culture.
[1] A protein having an amino acid sequence represented by SEQ ID NO: 2 or 4
[2] A protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4 with one or more amino acid residues deleted, substituted, or added, and having peptide synthesis activity
[3] A protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having peptide synthesis activity The method for producing a peptide according to claim 6, wherein the transformant is transformed with the DNA according to any of the following [1] to [3].
[1] DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 3
[2] A DNA consisting of a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1 or 3 and encoding a protein having peptide synthesis activity
[3] A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 3 under stringent conditions and encodes a protein having peptide synthesis activity