JP6086678B2 - Method for producing homoamino acid - Google Patents

Description

  The present invention relates to a method for producing an L-homoamino acid using a microorganism.

Homo amino acids are important compounds as pharmaceutical raw materials. For example, L-homophenylalanine has been used as a synthetic raw material for angiotensin synthase (ACE) inhibitors. Regarding the production method of L-homophenylalanine, DL-homophenylalanine is optically resolved as a diastereomeric salt (Patent Literature 1, Patent Literature 2), and amino acid aminotransferase is used to convert 2-oxo-4-phenylbutyric acid. A method for transferring an amino group (Patent Document 3), a method for reductive amination of 2-oxo-4-phenylbutyric acid using L-phenylalanine dehydrogenase (Non-Patent Document 1), a hydantoin to 5-benzylmethylhydantoin A method (Patent Literature 4, Patent Literature 5) and a chemical synthesis method (Patent Literature 6) in which a nuclease system (comprising hydantoin hydrolase and N-carbamyl amino acid hydrolase) is known are known.
However, these conventional methods all require chemical synthesis raw materials, and are not the best methods in terms of efficiency and environment.
On the other hand, a biosynthetic pathway for synthesizing 2-oxo-4-phenylbutyric acid from inexpensive glucose has been reported (Non-patent Document 2). The biosynthetic pathway is characterized by having an artificially mutated LeuA derived from S. coli.
Npun_F2464 derived from Nostoc punctiforme PCC73102 strain is a protein of unknown function that was revealed by genome sequencing of the strain.

JP-A-63-063646 JP-A 63-145256 US Pat. No. 6,146,859 Special registration 0272845 JP-A-6-334383 JP-A-10-287632

J. Org. Chem. (1990), Vol. 55, p5567-5571. ACS. Chem. Biol. (2012), Vol. 7 (4), p689-697.

  An object of the present invention is to provide an efficient method for producing L-homoamino acids using microorganisms.

The present invention relates to the following (1) to (6).
(1) Using a culture of a microorganism producing a protein selected from the group consisting of the following [1] to [3] or a treated product of the culture as an enzyme source, the enzyme source and the formula (I)

[Wherein, R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are preferably the same or different and each may have a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, an amino group or a substituent. A lower alkyl group, more preferably a hydrogen atom, a halogen atom or a hydroxyl group, which are the same or different, and more preferably R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are a hydrogen atom, and R ^1c is a hydroxyl group. R ^1a , R ^1b , R ^1d and R ^1e are hydrogen atoms, R ^1a is a halogen atom and R ^1b , R ^1c , R ^1d and R ^1e are hydrogen atoms, R ^1b is a halogen atom and R ^1a , R ^1c , R ^1d and R ^1e are hydrogen atoms, or R ^1c is a halogen atom and R ^1a , R ^1b , R ^1d and R ^1e are hydrogen atoms, most preferably R ^1a , R ^1b , R ^1c , R ^1d and R ^1e is a hydrogen atom, R ^1c is hydroxy group and R ^1a, R ^1b, R ^1d and R ^1e is a hydrogen atom, R ^1b is halogen atom and R ^1a, R ^1c, R ^1d and R ^1e water Atoms, or, R ^1c is halogen atom and R ^1a, R ^1b, R ^1d and R ^1e are hydrogen. The halogen atom is preferably a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine, more preferably fluorine or chlorine, and most preferably fluorine. The L-amino acid represented by formula (hereinafter also referred to as Compound I) is present in an aqueous medium, and the formula (II) is contained in the aqueous medium.

[Wherein R ^1a , R ^1b , R ^1c , R ^1d and R ^1e have the same meanings as described above] L-homoamino acid (hereinafter also referred to as Compound II) is produced and accumulated, and the medium A method for producing an L-homoamino acid, which comprises collecting the L-homoamino acid from
[1] a protein having the amino acid sequence represented by SEQ ID NO: 2 [2] consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2; A protein having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 2 [3] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and A protein (2) microorganism having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 2 is compared with the parent strain (III)

[Wherein R ^1a , R ^1b , R ^1c , R ^1d and R ^1e have the same ^{meanings as} described above] (hereinafter referred to as compound III) is represented by formula (IV).

[Wherein R ^1a , R ^1b , R ^1c , R ^1d, and R ^1e have the same ^{meaning as} described above] (hereinafter referred to as compound III conversion activity) .) Is the enhanced microorganism, The production method according to (1) above.
(3) The microorganism converts compound IV to formula (V) compared to the parent strain.

[Wherein R ^1a , R ^1b , R ^1c , R ^1d, and R ^1e have the same ^{meaning as} described above] (hereinafter referred to as compound IV conversion activity) .) Is the enhanced microorganism, The production method according to (1) or (2) above.
(4) The production method according to any one of (1) to (3), wherein R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are the same or different and are a hydrogen atom, a halogen atom or a hydroxyl group.
(5) The microorganism according to any one of the above (1) to (3) having the ability to produce and accumulate L-phenylalanine or L-tyrosine is cultured in a medium, and L-homophenylalanine or L- A method for producing L-homophenylalanine or L-homotyrosine, wherein homotyrosine is produced and accumulated, and L-homophenylalanine or L-homotyrosine is collected from the culture.
(6) The production method according to any one of (1) to (5) above, wherein the microorganism is a microorganism belonging to the genus Escherichia.

According to the present invention, Npun_F2464 derived from Nostoc punctiforme PCC73102 or a culture of a microorganism producing a homologous protein of the protein or a method for producing an L-homoamino acid using a treated product of the culture as an enzyme source, and L-phenylalanine or The microorganism having the ability to produce and accumulate L-tyrosine is cultured in a medium, and L-homophenylalanine or L-homotyrosine is produced and accumulated in the culture, and L-homophenylalanine or L-homotyrosine is produced from the culture. It is possible to provide a method for producing L-homophenylalanine or L-homotyrosine characterized by collecting

It is explanatory drawing which showed the biosynthetic pathway from L-phenylalanine to L-homophenylalanine.

1. Microorganisms used in the production method of the present invention Examples of the microorganisms used in the production method of the present invention include the microorganisms described in any of (1) to (4) below.
(1) Microorganism producing a protein selected from the group consisting of the following [1] to [3] The microorganism used in the production method of the present invention is selected from the group consisting of the following [1] to [3] protein,
[1] a protein having the amino acid sequence represented by SEQ ID NO: 2,
[2] Activity of a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having the amino acid sequence represented by SEQ ID NO: 2 A protein having the same activity as
[3] It consists of an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: A protein having the same activity as that of the protein having the amino acid sequence represented by 2;
Can be mentioned.
The activity of the protein having the amino acid sequence represented by SEQ ID NO: 2 is represented by the formula (VI)

[Wherein, R ^1a , R ^1b , R ^1c , R ^1d and R ^1e have the same ^{meanings as} described above] (hereinafter referred to as compound VI) is represented by formula (VII).

[Wherein R ^1a , R ^1b , R ^1c , R ^1d, and R ^1e have the same ^{meaning as} described above] (hereinafter referred to as compound VI conversion activity) .)
In the above, a protein having an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added, and having a compound VI conversion activity, can be obtained by using the site-directed mutagenesis method described below, for example, It can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 or DNA having the base sequence represented by SEQ ID NO: 1.
The number of amino acid residues to be deleted, substituted or added is 1 to 20, preferably 1 to 10, and most preferably 1 to 5.

The deletion, substitution or addition of 1 to 20 amino acids in the amino acid sequence represented by SEQ ID NO: 2 means that one or more amino acid residues are deleted or substituted at any position in the same sequence. Or it may be added.
Deletions, substitutions or additions may occur simultaneously, and the amino acids substituted or added may be natural or non-natural. 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, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Examples of amino acids that can be substituted with each other are shown below. Amino acids included in the same group can be substituted for 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 Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine The identity of the amino acid sequence and base sequence is determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 by Karlin and Altschul. , determined using the 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] It can be. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. Further, when an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are well known.
A protein comprising an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 is a protein having compound VI conversion activity, for example, DNA recombination A transformant expressing a protein whose activity is to be confirmed using a method is prepared, cultured in a medium, the culture is treated with ultrasonic waves, etc., compound VI is added to the treated product, and the synthesized compound VII is synthesized. The amount can be confirmed by measuring by HPLC.
Examples of the microorganism that produces a protein selected from the group consisting of the above [1] to [3] include a microorganism obtained by transforming a parental microorganism with a recombinant DNA having a DNA encoding the protein.
Examples of the microorganism include a microorganism that retains the DNA on the chromosomal DNA by transforming a parental microorganism with a recombinant DNA having a DNA encoding the protein, and the DNA outside the chromosomal DNA as plasmid DNA. Can be mentioned.
Microorganisms that possess the DNA on the chromosomal DNA of the parent strain, and microorganisms that possess the DNA as chromosomal DNA outside the chromosomal DNA are, for example, those that exist on the chromosomal DNA originally possessed by the microorganism. A method in which the gene cannot be amplified, but the DNA introduced by transformation is confirmed by PCR using a primer set capable of amplification, and the amount of transcription of the DNA by Northern blotting or the protein The production amount can be confirmed by Western blotting, for example, by comparing with the parent strain.
As a DNA encoding a protein selected from the group consisting of the above [1] to [3], specifically, a DNA selected from the group consisting of the following [4] to [7],
[4] DNA encoding the protein according to any one of [1] to [3] above,
[5] DNA having the base sequence represented by SEQ ID NO: 1,
[6] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a protein having compound VI conversion activity; and
[7] It comprises a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1, and is converted into Compound VI DNA encoding a protein having activity,
Can be mentioned.

  The term “hybridize” as used above means that the DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part thereof can be used as a probe for Northern or Southern blot analysis, or can be used as an oligonucleotide primer for PCR analysis. Examples of the DNA used as a probe include at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more. The DNA used as a primer is at least 10 bases, preferably 15 bases. The above DNA can be mentioned.

  Methods for DNA hybridization experiments are well known. For example, those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions are described in Molecular Cloning 2nd edition, 3rd edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press (1996). Can be done according to other standard textbooks.

  Furthermore, DNA that hybridizes under stringent conditions can also be obtained by following the instructions attached to a commercially available hybridization kit. Examples of commercially available hybridization kits include a random primed DNA labeling kit (Roche Diagnostics) that produces probes by the random prime method and performs hybridization under stringent conditions.

  The above stringent conditions are: DNA-immobilized filter and probe DNA are 50% formamide, 5 × SSC (750 mmol / l sodium chloride, 75 mmol / l sodium citrate), 50 mmol / l phosphoric acid. After incubation overnight at 42 ° C. in a solution containing sodium (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, X Conditions for washing the filter in SSC solution can be mentioned.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.
The DNA that can hybridize under the stringent conditions described above is, for example, a DNA comprising the base sequence represented by SEQ ID NO: 1 when calculated based on the above-mentioned parameters using BLAST, FASTA, etc. Mention may be made of DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more.
In this specification, the parent strain refers to the original strain that is the subject of genetic modification, transformation, and the like. The original strain to be transformed by gene transfer is also called a host strain.

Microorganism used in the present invention may be any microorganism, preferably a prokaryote, more preferably a bacterium, more preferably Escherichia (Escherichia) genus Enterobacter (Enterobacter) genus Pantoea (Pantoea) genus, Klebsiella (Klebsiella) genus Serratia (Serratia) genus Erwinia (Erwinia) genus Sarumonea (Salmonella) genus and Morganella (Morganella) microorganism belonging to a genus selected from the group consisting of the genus, particularly preferably microorganisms belonging to the genus Escherichia, most Preferably, Escherichia coli can be mentioned.
As Escherichia coli, a wild strain selected from the group consisting of Escherichia coli K-12 strain, B strain, W strain and B / r strain or a mutant strain thereof, preferably Escherichia coli XL1-Blue, Escherichia coli 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 W3110 Coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415, Escherichia coli ATCC9637, and the like.
(2) Microorganisms with enhanced compound III conversion activity compared to the parent strain
Examples of the microorganism used in the production method of the present invention include a microorganism having the above-mentioned (1) microorganism as a parent strain and having enhanced compound III conversion activity compared to the parent strain.
Microorganisms having enhanced compound III conversion activity compared to the parent strain include (a) obtained by modifying a gene encoding a protein having compound III conversion activity present on the chromosomal DNA of the parent strain, and i) compared to the parent strain. A microorganism in which the specific activity of the protein is enhanced, and ii) a microorganism in which the transcription amount of the gene or the production amount of the protein is increased compared to the parent strain, and (b) a recombinant DNA having a DNA encoding the protein By transforming the microorganism of the parent strain with, a microorganism having a copy number of the gene increased compared to the parent strain can be mentioned.
The protein having compound III conversion activity may be any protein as long as it has the activity. Specifically, a protein selected from the group consisting of the following [8] to [10],
[8] a protein having the amino acid sequence represented by SEQ ID NO: 4, 6 or 8,
[9] a protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, 6 or 8, and having a compound III conversion activity; and
[10] It consists of an amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 4, 6 or 8. And a protein having compound III conversion activity,
Can be mentioned.

It is described in Non-Patent Document 2 that a protein having the amino acid sequence represented by SEQ ID NO: 6 or 8 has compound III conversion activity.
In the above, a protein having an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added and having compound III conversion activity can be obtained by the same method as in 1 (1) above. it can. The number and position of amino acid residues to be deleted, substituted or added are the same as in 1 (1) above.
The protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, 6 or 8 is a protein having compound III conversion activity, For example, a transformant expressing a protein whose activity is to be confirmed is prepared using a DNA recombination method, cultured in a medium, and the culture is treated with ultrasonic waves, etc., and then compound III is added to the treated product. It can be confirmed by measuring the amount of Compound IV by HPLC.
The DNA encoding a protein having a compound III conversion activity may be any DNA as long as it encodes a protein having the activity. Specifically, the DNA is selected from the group consisting of the following [11] to [14]. DNA,
[11] DNA encoding the protein according to any one of [8] to [10] above,
[12] DNA having a base sequence represented by SEQ ID NO: 3, 5 or 7;
[13] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3, 5, or 7 and that encodes a protein having compound III conversion activity; and ,
[14] consisting of a base sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 3, 5 or 7. And a DNA encoding a protein having compound III conversion activity,
Can be mentioned.

The above description of “hybridize” and the method and conditions for DNA hybridization experiments are the same as in 1 (1) above.
The DNA that can hybridize under the above-mentioned stringent conditions includes, for example, a base sequence represented by SEQ ID NO: 3, 5, or 7 when calculated based on the above-mentioned parameters using BLAST, FASTA, etc. And DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
(3) Microorganism having enhanced compound IV conversion activity compared to the parent strain As a microorganism used in the production method of the present invention, a microorganism having enhanced compound IV conversion activity using the microorganism of (1) or (2) as a parent strain. Can be mentioned.
Microorganisms with enhanced compound IV conversion activity compared to the parent strain are (a) obtained by modifying a gene encoding a protein having compound IV conversion activity present on the chromosomal DNA of the parent strain, and i) compared with the parent strain. A microorganism in which the specific activity of the protein is enhanced, and ii) a microorganism in which the transcription amount of the gene or the production amount of the protein is increased compared to the parent strain, and (b) a recombinant DNA having a DNA encoding the protein By transforming the microorganism of the parent strain with, a microorganism having a copy number of the gene increased compared to the parent strain can be mentioned.
The protein having compound IV conversion activity may be any protein as long as it has the activity. Specifically, a protein selected from the group consisting of the following [15] to [17],
[15] a protein having the amino acid sequence represented by SEQ ID NO: 10 or 12,
[16] a protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 10 or 12, and having a compound IV conversion activity; and
[17] An amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 10 or 12, and A protein having compound IV conversion activity,
Can be mentioned.

It is described in Non-Patent Document 2 that the protein having the amino acid sequence represented by SEQ ID NO: 12 has compound IV conversion activity.
In the above, a protein having an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added and having compound IV conversion activity can be obtained by the same method as in 1 (1) above. it can. The number and position of amino acid residues to be deleted, substituted or added are the same as in 1 (1) above.
In the amino acid sequence represented by SEQ ID NO: 10 or 12, the protein comprising an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added is a protein having compound IV conversion activity, for example, DNA A recombinant compound is used to produce a transformant that expresses a protein whose activity is to be confirmed, cultured in a medium, and the culture is treated with ultrasonic waves, and then compound IV is added to the treated product to synthesize the compound. It can be confirmed by measuring the amount of V by HPLC.
The DNA encoding a protein having compound IV conversion activity may be any DNA as long as it encodes a protein having the activity, and specifically, is selected from the group consisting of the following [18] to [21] DNA,
[18] DNA encoding the protein according to any one of [15] to [17] above,
[19] DNA having the base sequence represented by SEQ ID NO: 9 or 11,
[20] DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 9 or 11, and encodes a protein having compound IV conversion activity; and
[21] A compound comprising a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 9 or 11. DNA encoding a protein having IV conversion activity,
Can be mentioned.

The above description of “hybridize” and the method and conditions for DNA hybridization experiments are the same as in 1 (1) above.
The DNA that can hybridize under the above-mentioned stringent conditions comprises, for example, the base sequence represented by SEQ ID NO: 9 or 11 when calculated based on the above-described parameters using BLAST, FASTA, etc. Mention may be made of DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with DNA.
(4) Microorganisms having the ability to produce and accumulate L-phenylalanine or L-tyrosine The microorganism used in the production method of the present invention has the ability to produce and accumulate L-phenylalanine or L-tyrosine (1) ) To (3).
The ability to produce and accumulate L-phenylalanine or L-tyrosine refers to the ability to produce and accumulate L-phenylalanine or L-tyrosine to such an extent that L-phenylalanine or L-tyrosine can be recovered from the culture when the microorganism used in the present invention is cultured in the medium. Say.

If the parent strain originally has the property, the microorganism having the ability may be a microorganism with enhanced properties, and if the parent strain does not have the property, the property is artificially changed. The microorganisms added to can be mentioned.
Examples of the microorganism include ATCC13281 and ATCC21699 as L-phenylalanine producing strains, and ATCC21652 as L-tyrosine producing strains.
2. Microorganism production method used in the production method of the present invention (1) Microorganism production method of 1 (1) above The microorganism of 1 (1) encodes a protein selected from the group consisting of [1] to [3] It can be constructed by transforming a parental microorganism with a recombinant DNA having the DNA to be transformed.

As the DNA encoding a protein selected from the group consisting of [1] to [3] of 1 (1) above, for example, a probe DNA that can be designed based on the base sequence represented by SEQ ID NO: 1 was used. Southern hybridization to a microorganism, preferably a microorganism belonging to the genus Nostock, more preferably a Nostoc punctiforme PCC7310 chromosomal DNA library, or a chromosomal DNA of the microorganism using a primer DNA that can be designed based on the base sequence PCR as a template [PCR Protocols, Academic Press (1990)].

In addition, a sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 with respect to various gene sequence databases. Based on the base sequence obtained by the search, a DNA encoding the protein can be obtained from the chromosomal DNA or cDNA library of the microorganism having the base sequence by the method described above.
The obtained DNA is cleaved as it is or with an appropriate restriction enzyme, and incorporated into a vector by a conventional method. After the obtained recombinant DNA is introduced into a host cell, a commonly used nucleotide sequence analysis method such as the dideoxy method [ Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or 3700 DNA analyzer (manufactured by Applied Biosystems) and other base sequence analyzers to determine the base sequence of the DNA can do.

The above vectors include pBluescriptII KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK (+) (Stratagene), pT7Blue ( Novagen), pCR II (Invitrogen) and pCR-TRAP (Gen Hunter).
As a method for introducing recombinant DNA, any method for introducing DNA into a host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electroporation method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

If the obtained DNA has a partial length as a result of determining the base sequence, the full-length DNA can be obtained by Southern hybridization or the like for a chromosomal DNA library using the partial length DNA as a probe. .
Furthermore, based on the determined DNA base sequence, the target DNA can be prepared by chemical synthesis using a 8905 type DNA synthesizer manufactured by Perceptive Biosystems.

Examples of DNA that can be obtained by the above method include DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or DNA having the base sequence represented by SEQ ID NO: 1.
Based on the DNA obtained by the above method, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared. In addition, a transformant with an improved production rate can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for expression in a host cell.

  A recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector. The recombinant DNA encodes a protein selected from the group consisting of [1] to [3] of 1 (1) above by, for example, transforming a microorganism belonging to the genus Escherichia of 1 (1) above. A microorganism obtained by transforming a parental microorganism with a recombinant DNA having DNA can be obtained.

As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA encoding the protein can be transcribed is used.
When a prokaryotic organism is used as a host cell, the recombinant DNA having the DNA is capable of autonomous replication in prokaryotes, and at the same time, a recombinant composed of a promoter, a ribosome binding sequence, the DNA, and a transcription termination sequence. It is preferably body DNA. A gene that controls the promoter may also be included.

  As an expression vector, for example, when Escherichia coli is used as a host cell, pColdI (manufactured by Takara Bio Inc.), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x (manufactured by New England Biolabs) ), PGEX-4T-1 (manufactured by GE Healthcare Biosciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pET -3 (manufactured by NOVAGEN), pKYP10 (Japanese Patent Laid-Open No. 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 (-) (Stratagene), pTrS30 [Escherichia coli JM109 / pTrS30 (Prepared from FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 200 7, Vol. 73, No. 20, p.6378-6385] pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.) PPA1 (Japanese Patent Laid-Open No. 63-233798) and the like.

Any promoter can be used as long as it functions in a host cell such as Escherichia coli, for example, trp promoter (P _trp ), lac promoter (P _lac ), P _L. promoter, P _R promoter and P _SE promoter, may be used promoters derived from Escherichia coli, phage and the like. When a microorganism belonging to the genus Bacillus is used as a parent strain, an SPO1 promoter, an SPO2 promoter, a penP promoter, etc. that function in Bacillus subtilis can also be used. Further, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a let I promoter can be used.

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 encoding the protein is bound to an expression vector, a transcription termination sequence is not necessarily required, but it is preferable to place the transcription termination sequence immediately below the structural gene.
An example of such a recombinant DNA is pSTV_F2464 described later.
In addition, the DNA obtained by the above method is also composed of [1] to [3] of the above 1 (1) by incorporating it into an arbitrary position of the chromosomal DNA of the parent strain by using the homologous recombination method. A microorganism obtained by transforming a parental microorganism with a recombinant DNA having a DNA encoding a protein selected from the group can be obtained.
As a method of replacing the target region on the chromosomal DNA of the parent strain by the homologous recombination method, a plasmid for homologous recombination that can be prepared by ligation with a plasmid DNA having a drug resistance gene that cannot autonomously replicate in the host cell to be introduced is used. As a method using homologous recombination frequently used in Escherichia coli, a method of introducing a base deletion, substitution or addition using a lambda phage homologous recombination system [Proc Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

In addition, a selection method that makes Escherichia coli susceptible to sucrose by Bacillus subtilis levanshuclase integrated on the chromosome together with the mutant DNA, and the incorporation of the wild-type rpsL gene into Escherichia coli having a mutant rpsL gene resistant to streptomycin. On the chromosomal DNA of the parent strain using a selection method that makes Escherichia coli susceptible to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)]. A strain in which the target region is replaced with the gene prepared by the above method can be obtained.

In addition to the above method, other gene replacement methods can be used as long as they can replace genes on the chromosomes of microorganisms.
It can be confirmed using the method 1 (1) that the microorganism obtained by the above method is a microorganism having a compound VI conversion activity.
(2) Method for constructing microorganism of 1 (2) above Among the microorganisms of 1 (2) above, (a) by modifying a gene encoding a protein having compound III conversion activity present on the chromosomal DNA of the parent strain The obtained i) microorganism having enhanced specific activity of the protein compared to the parent strain is, for example, a protein having compound III conversion activity of the parent strain using the microorganism constructed by the method of 2 (1) as a parent strain. The specific activity of can be increased by using a usual mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like.
Examples of the mutation treatment method include a method using N-methyl-N′-nitro-N-nitrosoguanidine (NTG) (Microbial Experiment Manual, 1986, p. 131, Kodansha Scientific), ultraviolet irradiation method, and the like. Can give.

  As a gene replacement method by recombinant DNA technology, after introducing a mutation into DNA encoding a protein having a compound III conversion activity by subjecting it to a mutation treatment using a mutation agent in a test tube or error-prone PCR, Examples of the method include substitution with the gene encoding the protein present on the chromosomal DNA of the parent strain using the homologous recombination method of 2 (1) above.

DNA encoding a protein having compound III conversion activity is, for example, a microorganism, preferably a microorganism belonging to the genus Escherichia, using a probe DNA that can be designed based on the base sequence of the DNA according to 1 (2) above, More preferably, it can be obtained by a method such as PCR using Escherichia coli chromosomal DNA as a template.
Examples of the DNA obtained as described above include DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 6 or 8, or DNA having the base sequence represented by SEQ ID NO: 5 or 7. be able to.

It can be confirmed using the method 1 (2) that the microorganism obtained by the above method is the target microorganism.
Among the microorganisms of the above 1 (2), (a) obtained by modifying a gene encoding a protein having a compound III conversion activity present on the chromosomal DNA of the parent strain, ii) the gene compared to the microorganism of the parent strain For example, the microorganism having the increased transcription amount or the production amount of the protein can be obtained by, for example, using the microorganism constructed by the above method 2 (1) as a parent strain, The protein production can be increased by using a normal mutation treatment method, a gene replacement method using recombinant DNA technology, or the like.
Examples of the mutation treatment method include the method 1 (1).
The gene replacement method by the recombinant DNA technique comprises a transcription regulatory region and a promoter region of a gene encoding a protein having a compound III conversion activity possessed by a parent strain, for example, a base sequence of 200 bp upstream, preferably 100 bp upstream of the start codon of the protein. A gene encoding a protein having a compound III conversion activity present on the chromosomal DNA of the parent strain, after introducing the mutation into the DNA by subjecting the DNA to a mutation treatment in vitro or error-prone PCR, etc .; A method of substitution using the homologous recombination method of (1) can be mentioned.

In addition, by replacing the promoter region of the gene encoding the protein having the compound III conversion activity of the parent strain with a known strong promoter sequence, a microorganism with improved production of the protein having the compound III conversion activity can be obtained from the parent strain. You can also
Such promoters, in the case of using a microorganism belonging to the genus Escherichia as the parent strain, trp promoter functional in Escherichia coli (P _trp), lac promoter (P _lac), P _L promoter, P _R promoter, P _SE promoter and the like When a microorganism belonging to the genus Bacillus is used as a parent strain for a promoter derived from Escherichia coli, phage, or the like, an SPO1, SPO2, or penP promoter that functions in Bacillus subtilis can be exemplified. In addition, artificially constructed promoters such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, a let I promoter and the like can also be mentioned.
The method according to 1 (2) above, wherein the microorganism obtained by the above method is a microorganism in which the transcription amount of a gene encoding a protein having compound III conversion activity or the production amount of the protein is increased as compared with the parent strain. Can be confirmed.
Among the microorganisms of the above 1 (2), (b) a gene encoding the protein rather than the parent strain by transforming the parent strain with a recombinant DNA having a DNA encoding a protein having a compound III conversion activity A microorganism having an increased copy number can be obtained by the following method.

The DNA encoding a protein having compound III conversion activity is, for example, a microorganism, preferably Nostock or Escherichia, using a probe DNA that can be designed based on the base sequence of the DNA according to 1 (2) above. Can be obtained by a method such as PCR using a chromosomal DNA of Nostoc punctiforme PCC7310 or Escherichia coli as a template.

As the DNA obtained as described above, for example, when the chromosome DNA of Nostoc punctiforme PCC7310 is used as a template, DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 4 or represented by SEQ ID NO: 3 When the DNA having the nucleotide sequence is Escherichia coli chromosomal DNA as a template, the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 6 or 8 or the base represented by SEQ ID NO: 5 or 7 Mention may be made of DNA having a sequence.

  Based on the DNA encoding the protein having compound III conversion activity obtained by the above method, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared as necessary. In addition, a transformant with an improved production rate can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for expression in a host cell.

A recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector. By transforming a microorganism constructed by the above method 2 (1) with the recombinant DNA, a microorganism having an increased copy number of the gene encoding the protein than the parent strain can be obtained.
As the expression vector, a vector that contains a promoter at a position capable of transcription of DNA encoding a protein having L-asparagine excretion activity, which can autonomously replicate in the host cell or can be integrated into a chromosome, is used. .

When a prokaryotic organism is used as a host cell, the recombinant DNA having the DNA is capable of autonomous replication in prokaryotes, and at the same time, a recombinant composed of a promoter, a ribosome binding sequence, the DNA, and a transcription termination sequence. It is preferably body DNA. A gene that controls the promoter may also be included.
As an expression vector, for example, when Escherichia coli is used as a host cell, the expression vector 2 (1) can be mentioned.

Examples of the promoter when using the expression vector include the promoter described in 2 (1) above.
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 a recombinant DNA in which a DNA encoding a protein having compound III conversion activity is bound to an expression vector, a transcription termination sequence is not necessarily required, but it is preferable to place the transcription termination sequence immediately below the structural gene.

An example of such a recombinant DNA is pSTV_F2458 described later.
In addition, by using the homologous recombination method described in 2 (1) above, the DNA encoding the protein having the compound III conversion activity obtained by the above method can be incorporated into any position of the chromosomal DNA from the parent strain. In addition, a microorganism having an increased number of copies of the gene encoding the protein can be obtained.
It can be confirmed by using the method 1 (2) that the microorganism obtained by the above method has an increased number of copies of a gene encoding a protein having a compound III conversion activity as compared with the parent strain. .
(3) Method for constructing microorganism of 1 (3) above Among the microorganisms of 1 (3) above, (a) by modifying a gene encoding a protein having a compound IV conversion activity present on the chromosomal DNA of the parent strain The obtained i) the microorganism having a higher specific activity of the protein than the parent strain is obtained by, for example, converting the parent strain as a parent strain to the compound constructed by the above method 2 (1) or (2). The specific activity of a protein having activity can be obtained by enhancing it using a normal mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like.
Examples of the mutation treatment method include the above-described method 2 (2).

  As a gene replacement method by recombinant DNA technology, after introducing a mutation into DNA encoding a protein having a compound IV conversion activity by subjecting it to a mutation treatment using a mutation agent in a test tube or error-prone PCR, Examples of the method include substitution with the gene encoding the protein present on the chromosomal DNA of the parent strain using the homologous recombination method of 2 (1) above.

DNA encoding a protein having compound IV conversion activity is, for example, a microorganism, preferably a microorganism belonging to the genus Escherichia, using a probe DNA that can be designed based on the base sequence of the DNA according to 1 (2) above, More preferably, it can be obtained by a method such as PCR using Escherichia coli chromosomal DNA as a template.
Examples of the DNA obtained as described above include DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 12 or DNA having the base sequence represented by SEQ ID NO: 11.

It can be confirmed using the method 1 (3) that the microorganism obtained by the above method is the target microorganism.
Among the microorganisms of the above 1 (3), (a) obtained by modifying a gene encoding a protein having a compound IV conversion activity present on the chromosomal DNA of the parent strain, ii) the gene as compared with the microorganism of the parent strain For example, a microorganism having an increased amount of transcription or production of the protein can be obtained by, for example, using a microorganism produced by the above method 2 (1) or (2) as a parent strain and a gene of a protein having compound IV conversion activity of the parent strain. The amount of transcription or the production amount of the protein can be obtained by enhancing using a usual mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like.
Examples of the mutation treatment method include the method 1 (1).
The gene replacement method by the recombinant DNA technique comprises a transcription regulatory region and a promoter region of a gene encoding a protein having a compound IV conversion activity possessed by a parent strain, for example, a base sequence of 200 bp upstream, preferably 100 bp upstream of the start codon of the protein. A gene encoding a protein having a compound IV conversion activity present on the chromosomal DNA of the parent strain after introducing the mutation into the DNA by subjecting the DNA to a mutation treatment in a test tube or error-prone PCR or the like; A method of substitution using the homologous recombination method of (1) can be mentioned.

In addition, by replacing the promoter region of the gene encoding the protein having the compound IV conversion activity of the parent strain with a known strong promoter sequence, a microorganism having an improved production amount of the protein having the compound IV conversion activity can be obtained from the parent strain. You can also
As such a promoter, when a microorganism belonging to the genus Escherichia is used as a parent strain, the promoter described in 2 (2) above can be mentioned.
The method according to 1 (3) above, wherein the microorganism obtained by the above method is a microorganism in which the transcription amount of a gene encoding a protein having a compound IV conversion activity or the production amount of the protein is increased as compared with the parent strain. Can be confirmed.
Among the microorganisms of the above 1 (3), (b) a gene encoding the protein rather than the parent strain by transforming the parental microorganism with a recombinant DNA having a DNA encoding a protein having a compound IV conversion activity A microorganism having an increased copy number can be obtained by the following method.

The DNA encoding the protein having compound IV conversion activity is, for example, a microorganism, preferably Nostock or Escherichia, using a probe DNA that can be designed based on the base sequence of the DNA according to 1 (3) above. Can be obtained by a method such as PCR using a chromosomal DNA of Nostoc punctiforme PCC7310 or Escherichia coli as a template.

As the DNA obtained as described above, for example, when the chromosome DNA of Nostoc punctiforme PCC7310 is used as a template, DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 10 or represented by SEQ ID NO: 9 DNA having the base sequence represented by SEQ ID NO: 11 or DNA having the base sequence represented by SEQ ID NO: 11 when Escherichia coli chromosomal DNA is used as a template Can be mentioned.

  Based on the DNA encoding the protein having compound IV conversion activity obtained by the above method, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared as necessary. In addition, a transformant with an improved production rate can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for expression in a host cell.

A recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector. Obtaining a microorganism having an increased copy number of the gene encoding the protein than the parent strain by transforming the microorganism constructed by the method of 2 (1) or (2) with the recombinant DNA Can do.
As the expression vector, a vector that contains a promoter at a position where it can autonomously replicate in the host cell or can be integrated into a chromosome and can transcribe a DNA encoding a protein having compound IV conversion activity is used.

When a prokaryotic organism is used as a host cell, the recombinant DNA having the DNA is capable of autonomous replication in prokaryotes, and at the same time, a recombinant composed of a promoter, a ribosome binding sequence, the DNA, and a transcription termination sequence. It is preferably body DNA. A gene that controls the promoter may also be included.
As an expression vector, for example, when Escherichia coli is used as a host cell, the expression vector 2 (1) can be mentioned.

Examples of the promoter when using the expression vector include the promoter described in 2 (1) above.
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 a recombinant DNA in which a DNA encoding a protein having a compound IV conversion activity is bound to an expression vector, a transcription termination sequence is not necessarily required, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

An example of such a recombinant DNA is pSTV_F2457 described later.
In addition, by using the homologous recombination method described in 2 (1) above, the DNA encoding the protein having the compound IV conversion activity obtained by the above method can be incorporated into any position of the chromosomal DNA from the parent strain. In addition, a microorganism having an increased number of copies of the gene encoding the protein can be obtained.
It can be confirmed using the method 1 (3) that the microorganism obtained by the above method has an increased number of copies of a gene encoding a protein having a compound IV conversion activity as compared with the parent strain. .
(4) The method for producing a microorganism according to 1 (4) above, wherein the microorganism has the ability to produce and accumulate L-phenylalanine or L-tyrosine according to 1 (4) above. The microorganism may be any microorganism as long as it has the ability, and when the strain itself isolated from nature has the ability, the strain itself may be the desired L-amino acid by a known method. A microorganism artificially imparted to the microorganism according to any one of the above 1 (1) to (3) can be mentioned.

As the known method,
(A) a method for mitigating or releasing at least one of the mechanisms controlling amino acid biosynthesis;
(B) a method for enhancing expression of at least one enzyme involved in amino acid biosynthesis;
(C) a method for increasing the number of copies of at least one enzyme gene involved in amino acid biosynthesis;
(D) a method of weakening or blocking at least one metabolic pathway branching from an amino acid biosynthetic pathway to a metabolite other than the amino acid; and (e) a cell having a higher resistance to an amino acid analog than a wild-type strain. The method of selecting a strain | stump | stock etc. can be mention | raise | lifted, The said well-known method can be used individually 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 (b) are described in, for example, Agric. Biol. Chem., 43, 105-111 (1979) and J. Bacteriol., 110, 761-763 (1972), etc. 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 (e), 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. A microorganism having the ability to produce and accumulate various amino acids can be prepared with reference to the above documents and the like.

  Furthermore, for a method for preparing a microorganism having the ability to produce and accumulate amino acids by any one of (a) to (e) above 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, Hiroshi Aida et al. (1986) In addition to the above, methods for preparing microorganisms having the ability to produce and accumulate specific amino acids are disclosed in JP 2003-164297, Agric. Biol. Chem., 39, 153-160 (1975), Agric. Biol. Chem. , 39, 1149-1153 (1975), JP 58-13599, J. Gen. Appl.Microbiol., 4, 272-283 (1958), JP 63-94985, Agric. Biol. Chem., 37, 2013-2023 (1973), WO97 / 15673, JP-A-56-18596, JP-A-56-144092, and Special Table 2003-511086, etc. Production of amino acids, the microorganism having an ability to accumulate can be prepared by.

Examples of L-phenylalanine producing bacteria that can be prepared by the above method include Escherichia coli transformed with a DNA encoding a phenylalanine biosynthesis enzyme (JP-A-5-344881, JP-A-5-4881). 236947).
Specific examples include ATCC13281 and ATCC21699 as L-phenylalanine producing strains and ATCC21652 as L-tyrosine producing strains.

The strain represented by the above ATCC number can be obtained from the American Type Culture Collection (USA).
3. Production method of L-homoamino acid of the present invention (1) Production method of L-homoamino acid using a culture of microorganism or treated product of the culture as an enzyme source 2) A culture of microorganisms that can be produced by the method of (1) to (4) or a treated product of the culture is used as an enzyme source, and the enzyme source and compound I that is an L-amino acid are present in an aqueous medium, A method for producing L-homoamino acid, which comprises producing and accumulating compound II which is an L-homoamino acid in an aqueous medium, and collecting the L-homoamino acid from the medium, can be mentioned.

The culture of microorganisms used in the production method can be obtained by culturing microorganisms that can be produced by the above methods 2 (1) to (4) by the following method.
The medium used in the production method of the present invention may be either a synthetic medium or a natural medium as long as it contains nutrients necessary for the growth of the microorganism to be used, such as a carbon source, a nitrogen source, and an inorganic salt.
The carbon source may be any carbon source that can be assimilated by the microorganism used, for example, sugars such as glucose, molasses, fructose, sucrose, maltose, starch hydrolysate, alcohols such as ethanol and glycerol, Examples thereof include organic acids such as acetic acid, lactic acid, and succinic acid.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, nitrogen compounds such as urea and amines, meat extract, yeast extract, corn steep liquor, peptone, soybean Nitrogen-containing organic substances such as hydrolysates can be used.
As the inorganic salt, potassium hydrogen phosphate, potassium hydrogen phosphate, ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium carbonate and the like can be used.

In addition, trace nutrient sources such as biotin, thiamine, nicotinamide, and nicotinic acid can be added as necessary. These micronutrient sources can be substituted with medium additives such as meat extract, corn steep liquor, casamino acid and the like.
Furthermore, if necessary, a substance required for growth by a microorganism to be cultured (for example, a required amino acid in the case of an amino acid-requiring microorganism) can be added.

  The culture is performed under aerobic conditions such as shaking culture and deep aeration stirring culture. The culture temperature is 20 to 50 ° C, preferably 20 to 30 ° C, more preferably 25 to 30 ° C. The culture is performed while maintaining the pH of the medium in the range of 5 to 11, preferably in the vicinity of neutrality of 6 to 9. The pH of the medium is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, a pH buffer solution, or the like.

The culture time is 5 hours to 6 days, preferably 16 hours to 3 days.
In addition, as a processed product of a microorganism culture used in the production method, a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging or filtering the culture, Such as a dried product of the cells, a freeze-dried product of the cells, a surfactant-treated product of the cells, a solvent-treated product of the cells, an enzyme-treated product of the cells, and an immobilized product of the cells As a source of enzyme, containing a viable cell body having the same function as that of the culture, an ultrasonically treated product of the cell, a mechanically ground product of the cell, and the treated cell Examples thereof include crude enzyme extracts obtained.

In the production method, Compound I, which is an L-amino acid used as a substrate, is added to an aqueous medium at the beginning or during the reaction so as to have a concentration of 0.1 to 500 g / L, preferably 0.2 to 200 g / L.
The amount of the culture of the microorganism used as the enzyme source or the treated product of the culture varies depending on the specific activity of the enzyme source, etc., for example, 5 to 1000 mg as a wet cell weight per 1 mg of the L-amino acid used as a substrate, Preferably 10 to 400 mg is added.
The aqueous medium may be an aqueous medium of any component and composition as long as it does not inhibit the formation reaction of the compound II that is an L-homoamino acid. For example, water, phosphate, carbonate, acetate, borate And buffer solutions 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. In addition, the culture supernatant of the microorganism or transformant culture used for the enzyme source can also be used as an aqueous medium.

The formation reaction of compound II which is an L-homoamino acid is carried out in an aqueous medium at pH 5-11, preferably pH 6-10, 20-50 ° C., preferably 25-45 ° C., 2-150 hours, preferably 6-120. Do time.
The compound II, which is an L-homoamino acid, in the aqueous medium can be usually collected by combining ion exchange resin method, precipitation method and other known methods. In addition, when the L-homoamino acid accumulates in the microbial cells, for example, the microbial cells are disrupted by ultrasonic waves and the microbial cells are removed by centrifugation, and the supernatant is obtained by ion exchange resin method or the like. Compound II, which is an L-homo amino acid, can be collected.
In the production method, Compound I which is an L-amino acid used as a substrate and Compound II which is an L-homoamino acid produced can be analyzed and quantified by the following method.
When using HPLC, the supernatant obtained by centrifuging the above-mentioned aqueous medium at 13,000 rpm for 30 minutes is obtained using Inertosil ODS-3 4.6 x 150 mm (GL sciences Inc., 4.6 x 150 mm). It can be analyzed and quantified. As the mobile phase, a mobile phase A of water containing trifluoroacetic acid having a final concentration of 0.1% and a mobile phase B of methanol containing trifluoroacetic acid having a final concentration of 0.1% can be used. The flow rate of the mobile phase is 1.0 mL / min, the mixing ratio of mobile phase A and mobile phase B is a gradient of 75:25 to 65:35 for 0-15 minutes, and 65:35 to 10:90 for 15-16 minutes. The slope, 16-18 minutes, can be constant at 10:90, 18-19 minutes can be varied from 10:90 to 75:25, and 19-25 minutes can be constant at 75:25. UV absorption at a column temperature of 50 ° C. and 220 nm can be measured.
When LC-ESI-MS is used, Inertosil ODS-3 4.6 x 150 mm (GL sciences Inc., 4.6 x 150 mm) is used as the HPLC separation column, and LC / MS / MS 3200Q TRAP ( A sample can be analyzed by a system combined with (ABC). As the mobile phase conditions, the same conditions as those used in the above HPLC analysis can be used.
(2) Method for Producing L-Homophenylalanine or L-Tyrosine by Fermentation Method As a method for producing L-homophenylalanine or L-tyrosine of the present invention, the above-mentioned having the ability to produce and accumulate L-phenylalanine or L-tyrosine Microorganisms that can be produced by the method 2 (4) are cultured in a medium, L-homophenylalanine or L-homotyrosine is produced and accumulated in the culture, and L-homophenylalanine or L-homotyrosine is collected from the culture. And a method for producing L-homophenylalanine or L-homotyrosine, which is characterized by the above.

L-homophenylalanine refers to an L-homoamino acid in which R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are represented by hydrogen atoms in the compound II which is an L-homoamino acid.
L-homotyrosine refers to an L-homoamino acid in the compound II which is an L-homoamino acid, wherein R ^1c is a hydroxyl group and R ^1a , R ^1b , R ^1d and R ^1e are hydrogen atoms.
Examples of the culture method of the microorganism include the culture method described in 3 (1) above.
Collection of L-homophenylalanine or L-homotyrosine from the culture can be usually carried out by a combination of ion exchange resin method, precipitation method and other known methods. In addition, when L-homophenylalanine or L-homotyrosine accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves and the like, and the microbial cells are removed by centrifugation. Can collect L-homophenylalanine or L-homotyrosine.
In the production method, L-phenylalanine or L-tyrosine used as a substrate and L-homophenylalanine or L-homotyrosine produced can be analyzed and quantified using the method of 3 (1) above.

  Examples of the present invention are shown below, but the present invention is not limited to these examples.

Construction of transformant transformed with DNA encoding protein having compound VI conversion activity, compound III conversion activity and compound IV conversion activity (1) Construction of W3110 / pTrc_F2464-F2458 / pSTV_F2457
PCR reaction was carried out using the chromosomal DNA of Nostoc punctiforme PCC73102 strain (hereinafter referred to as PCC73102 strain) as a template and oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 13 and 14 as primer sets. The amplified product obtained by the PCR is cleaved with NcoI and BamHI and ligated to an expression vector pTrc99A (manufactured by Invitrogen) cleaved with NcoI and BamHI, thereby expressing an expression plasmid pTrc99A_F2464 having the base sequence represented by SEQ ID NO: 1. Got.

PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 15 and 16 as primer sets. The amplified product obtained by the PCR was cleaved with BamHI and HindIII, and ligated to pTrc99A_F2464 cleaved with BamHI and HindIII to obtain an expression plasmid pTrc_F2464-F2458 having the base sequence represented by SEQ ID NOs: 1 and 3. .
PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 17 and 18 as primer sets. An amplification product (hereinafter referred to as F2457-BP fragment) obtained by the PCR was cleaved with BamHI and PstI and ligated to pTrc99A cleaved with BamHI and PstI to obtain an expression plasmid pTrc99A-F2457.
PCR reaction was performed using the DNA of the expression plasmid pTrc99A-F2457 as a template and oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 19 and 18 as a primer set. The amplified product obtained by the PCR was cleaved with SacI and PstI, and ligated to pSTV29 (manufactured by Takara Bio Inc.) cleaved with SacI and PstI, whereby the expression plasmid pSTV− having the base sequence represented by SEQ ID NO: 9 F2457 was obtained.
Escherichia coli W3110 strain was transformed with the expression plasmids pTrc_F2464-F2458 and pSTV-F2457 obtained above to obtain W3110 / pTrc_F2464-F2458 / pSTV_F2457.
(2) Construction of W3110 / pTrc_F2464-F2457 / pSTV_F2458 The following expression system was constructed by changing the arrangement of DNAs having the nucleotide sequences represented by SEQ ID NOs: 1, 3 and 9 by the same method as described above.
The above F2457-BP fragment was cleaved with BamHI and PstI and ligated to pTrc99A_F2464 cleaved with BamHI and PstI to obtain an expression plasmid pTrc99A_F2464-F2457 having the nucleotide sequences represented by SEQ ID NOs: 1 and 9.
PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 20 and 21 as primer sets. An amplification product (hereinafter referred to as F2458-EK fragment) obtained by the PCR was cleaved with EcoRI and KpnI and ligated to pTrc99A cleaved with EcoRI and KpnI to obtain an expression plasmid pTrc99A-F2458-2.
A PCR reaction was performed using the DNA of the expression plasmid pTrc99A-F2458-2 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 19 and 21 as primer sets. The amplified product obtained by the PCR was cleaved with SacI and KpnI and ligated to pSTV29 cleaved with SacI and KpnI to obtain an expression plasmid pSTV-F2458 having the base sequence represented by SEQ ID NO: 3.
Escherichia coli W3110 was transformed with the expression plasmids pTrc_F2464-F2457 and pSTV-F2458 obtained above to obtain W3110 / pTrc_F2464-F2457 / pSTV_F2458.
(3) Creation of W3110 / pTrc_F2457-F2464 / pSTV_F2458
PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 22 and 23 as a primer set. The amplified product obtained by the PCR was cleaved with NcoI and EcoRI, and ligated to the expression vector pTrc99A cleaved with NcoI and EcoRI to obtain an expression plasmid pTrc99A_F2457-2 having the base sequence represented by SEQ ID NO: 9. .

PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 24 and 14 as primer sets. The amplified product (hereinafter referred to as F2464-KB fragment) obtained by the PCR is cleaved with KpnI and BamHI, and ligated to pTrc99A_F2457-2 cleaved with KpnI and BamHI. An expression plasmid pTrc_F2457-F2464 having a base sequence was obtained.
Escherichia coli W3110 was transformed with the expression plasmids pTrc_F2457-F2464 and pSTV-F2458 obtained above to obtain W3110 / pTrc_F2457-F2464 / pSTV_F2458.
(4) Construction of W3110 / pTrc_F2457-F2458 / pSTV_F2464 The above F2458-EK fragment is cleaved with EcoRI and KpnI, and ligated to pTrc99A_F2457-2 cleaved with EcoRI and KpnI. An expression plasmid pTrc_F2457-F2458 having a base sequence was obtained.
PCR reaction was performed using the chromosomal DNA of PCC73102 strain as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 25 and 14 as primer sets. The amplification product obtained by the PCR was cleaved with NcoI and BamHI, and ligated to pTrc99A cleaved with NcoI and BamHI to obtain an expression plasmid pTrc99A-F2464-2.
PCR reaction was carried out using the DNA of the expression plasmid pTrc99A-F2464-2 as a template and oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 19 and 14 as primer sets. The amplified product obtained by the PCR was cleaved with SacI and BamHI and ligated to pSTV29 cleaved with SacI and BamHI to obtain an expression plasmid pSTV-F2464 having the base sequence represented by SEQ ID NO: 1.
Escherichia coli W3110 strain was transformed with the expression plasmids pTrc_F2457-F2458 and pSTV-F2464 obtained above to obtain W3110 / pTrc_F2457-F2458 / pSTV_F2464.
(5) Construction of W3110 / pTrc_F2458-F2464 / pSTV_F2457 The F2458-EK fragment obtained above is cleaved with EcoRI and KpnI, and ligated to pTrc99A cleaved with EcoRI and KpnI, whereby the base represented by SEQ ID NO: 3 An expression plasmid pTrc99A_F2458-2 having the sequence was obtained.
The F2464-KB fragment obtained above was cleaved with KpnI and BamHI and ligated to pTrc99A_F2458-2 cleaved with KpnI and BamHI, whereby the expression plasmid pTrc_F2458-F2464 having the nucleotide sequence represented by SEQ ID NOs: 1 and 3 was obtained. Got.
Escherichia coli W3110 strain was transformed with the expression plasmids pTrc_F2458-F2464 and pSTV-F2457 obtained above to obtain W3110 / pTrc_F2458-F2464 / pSTV_F2457.

Production of L-homoamino acid with added substrate (I)
Among the transformants obtained by the above operation, W3110 / pTrc_F2464-F2458 / pSTV_F2457, W3110 / pTrc_F2458-F2464 / pSTV_F2457, W3110 / pTrc_F2464-F2457 / pSTV_F2458, W3110 / pTrc_F2457-V2F_4582 pSTV_F2464 was cultured on an LB plate at 30 ° C. overnight, inoculated into a large test tube containing 5 mL of LB medium, and cultured with shaking at 30 ° C. for 12 hours. If necessary, 100 mg / L ampicillin and 25 mg / L chloramphenicol were added. Test tube production medium [glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, casamino acid 5 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, potassium dihydrogen phosphate 14 g / L, hydrogen phosphate Dipotassium 16 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, 1 g / L L-phenylalanine, pH 7 with sodium hydroxide solution After adjusting to .2, glucose and magnesium sulfate heptahydrate aqueous solution were autoclaved separately, inoculated with 0.1 mL into a large test tube containing 5 mL of “mixed after cooling”, cultured at 30 ° C. for 6 hours, and finished. IPTG was added so as to have a concentration of 1 mM, followed by shaking culture at 30 ° C. for 48 hours.
After completion of the culture, the culture solution was appropriately diluted and then centrifuged, and the L-homophenylalanine concentration in the supernatant was quantified by HPLC. L-homophenylalanine was detected and quantified by UV absorption analysis at a wavelength of 220 nm. The results are shown in Table 1.

Production of L-homoamino acid by fermentation method Transformants W3110 / pTrc_F2464-F2458 / pSTV_F2457 and W3110 / pTrc_F2457-F2458 / pSTV_F2464 obtained in Example 1 were converted to a plasmid conferring L-phenylalanine producing ability; pMW219_Ptrp-pheA ( 330) -aroG (P150I) (JP-A-5-344881, JP-A-5-236947) and transformed into W3110 / pTrc_F2464-F2458 / pSTV_F2457 / pMW219_Ptrp-pheA (330) -aroG (P150I) and W3110 / pTrc_F2457-F2458 / pSTV_F2464 / pMW219_Ptrp-pheA (330) -aroG (P150I) was obtained. Among the transformants obtained by the above operation, W3110 / pTrc_F2457-F2458 / pSTV_F2464 / pMW219_Ptrp-pheA (330) -aroG (P150I) and W3110 / pTrc_F2457-F2458 / pSTV_F2464 / pMW219_Ptrp-pheA (150) -aroG (330) -aroG ) Was cultured overnight on an LB plate at 30 ° C., inoculated into a large test tube containing 5 mL of LB medium, and cultured with shaking at 30 ° C. for 12 hours. As needed, 100 mg / L ampicillin, 25 mg / L chloramphenicol, 50 mg / L kanamycin were added. Test tube production medium not containing L-phenylalanine [glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, casamino acid 5 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, potassium dihydrogen phosphate 14 g / L, dipotassium hydrogen phosphate 16 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, pH 7 with sodium hydroxide solution. After adjusting to 2, the glucose and magnesium sulfate heptahydrate aqueous solution was autoclaved separately, cooled and then mixed with 0.1 mL in a large test tube containing 5 mL, cultured at 30 ° C. for 6 hours, and then the final concentration IPTG was added so as to be 1 mM, followed by shaking culture at 30 ° C. for 48 hours.
After completion of the culture, the culture solution was appropriately diluted and then centrifuged, and the L-homophenylalanine concentration in the supernatant was quantified by HPLC. L-homophenylalanine was detected and quantified by UV absorption analysis at a wavelength of 220 nm. The results are shown in Table 2.

Production of L-homoamino acid with added substrate (II)
Among the transformants obtained in Example 1, W3110 / pTrc_F2464-F2458 / pSTV_F2457 was cultured overnight at 30 ° C. on an LB plate, and inoculated into a large test tube containing 5 mL of LB medium. The culture was shaken at 30 ° C. for 12 hours. If necessary, 100 mg / L ampicillin and 25 mg / L chloramphenicol were added. Test tube production medium supplemented with 1 g / L of m-fluoro-DL-phenylalanine, p-fluoro-DL-phenylalanine, L-tyrosine [glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, casamino acid 5 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, potassium dihydrogen phosphate 14 g / L, dipotassium hydrogen phosphate 16 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L L, manganese sulfate pentahydrate 10 mg / L, 1 g / L L-phenylalanine derivative, adjusted to pH 7.2 with sodium hydroxide solution, glucose and magnesium sulfate heptahydrate aqueous solution were autoclaved separately and cooled Inoculate 0.1 mL into a large test tube containing 5 mL of post-mix], incubate at 30 ° C. for 6 hours, add IPTG to a final concentration of 1 mM, and culture at 30 ° C. for 48 hours with shaking. . The structure of the product was confirmed by analysis using HPLC and HPLC-ESI-MS. The HPLC analysis was performed by the same method as in Example 4 or a similar method. The results are shown in Table 3.

  According to the present invention, L-homoamino acids can be produced efficiently.

SEQ ID NO: 13—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 14—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 15—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 16—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 18—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 19—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 20—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 21—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 22—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 23—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 24—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 25—Description of Artificial Sequence: Synthetic DNA

Claims (4)

Producing a protein selected from the group consisting of [1] to [3] below, a protein selected from the group consisting of [4] to [6], and a protein selected from the group consisting of [7] to [9] Using a culture of microorganisms or a treated product of the culture as an enzyme source, the enzyme source and formula (I)

[Wherein, R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are the same or different and represent a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, an amino group, or a lower alkyl group which may have a substituent. L-amino acid represented by the formula (II) is present in an aqueous medium.

[Wherein, R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are as defined above] are generated and accumulated, and the L-homo amino acid is collected from the medium A process for producing an L-homoamino acid characterized by the above.
[1] a protein having the amino acid sequence represented by SEQ ID NO: 2 [2] consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2; A protein having the same activity as that of the protein having the amino acid sequence represented by SEQ ID NO: 2 [3] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and A protein having the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 2
[4] A protein having the amino acid sequence represented by SEQ ID NO: 4
[5] The amino acid sequence represented by SEQ ID NO: 4 consists of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added, and has the formula (III)

[Wherein R ^1a , R ^1b , R ^1c , R ^1d and R ^1e are as defined above]

[Wherein R ^1a , R ^1b , R ^1c , R ^1d, and R ^1e have the same ^{meaning as} described above], and a protein having an activity to convert to a compound (hereinafter referred to as compound III conversion activity).
[6] A protein comprising an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 4, and having a compound III conversion activity
[7] A protein having the amino acid sequence represented by SEQ ID NO: 10 or 12
[8] The amino acid sequence represented by SEQ ID NO: 10 or 12, consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added, and the formula (IV)

[Wherein R ^1a , R ^1b , R ^1c , R ^1d and R ^1e have the same ^{meanings as} described above]

[Wherein R ^1a , R ^1b , R ^1c , R ^1d, and R ^1e have the same ^{meaning as} described above], and a protein having an activity to convert to a compound (hereinafter referred to as compound IV conversion activity).
[9] A protein comprising an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 10 or 12, and having a compound IV conversion activity R ^1a , R ^1b , R ^1c , R ^1d And R ^1e Are the same or different and are a hydrogen atom, a halogen atom, and a hydroxyl group, The manufacturing method of Claim 1. Furthermore, the microorganism according to claim 1 having the ability to produce and accumulate L-phenylalanine or L-tyrosine is cultured in a medium, and L-homophenylalanine or L-homotyrosine is produced and accumulated in the culture, A method for producing L-homophenylalanine or L-homotyrosine, wherein L-homophenylalanine or L-homotyrosine is collected from the product. The production method according to any one of claims 1 to 3, wherein the microorganism is a microorganism belonging to the genus Escherichia.