Method for producing dipeptides

Abstract

The present invention addresses the problem of providing: a protein having improved substrate specificity and having dipeptide synthetic activity; and a method for producing a desired dipeptide, the method using said protein or a microorganism capable of producing said protein to reduce by-product dipeptides other than the desired dipeptide and to efficiently produce the desired dipeptide. The present invention provides a protein comprising an amino-acid sequence represented by SEQ ID NO: 2 or an amino acid sequence of a mutant or a homolog of the protein comprising the amino acid sequence represented by SEQ ID NO: 2, wherein amino acid residues corresponding to one or more amino acid residues at positions selected from the group consisting of position 107, position 108, and position 110 are substituted with other amino acid residues. Furthermore, the present invention is capable of efficiently producing dipeptides by utilizing a microorganism that produces said protein.

Description

[Technical Field]

[0001] The present invention relates to a protein having dipeptide synthesis activity with improved substrate specificity, and a method for efficiently producing a target dipeptide while reducing by-product dipeptides other than the target dipeptide by using the protein or a microorganism capable of producing the protein. [Background technology]

[0002] Microorganisms belonging to the genus Bacillus have been reported to possess L-amino acid α-ligase, which synthesizes dipeptides from ATP and two L-amino acids bound at the α-carboxyl group (Patent Document 1 and Non-Patent Document 1). L-amino acid α-ligase is extremely useful for efficient dipeptide production because it can synthesize dipeptides without the need for amino acid modification or special coenzymes. However, L-amino acid α-ligase has low substrate specificity, resulting in the production of dipeptides other than the desired dipeptide as by-products. For example, Non-Patent Document 2 discloses a method for producing L-alanyl-L-glutamine by transforming Escherichia coli with DNA encoding YwfE, a protein with L-amino acid α-ligase activity derived from Bacillus subtilis. However, it has been reported that in this process, L-alanyl-L-alanine is significantly produced as a by-product dipeptide in addition to L-alanyl-L-glutamine.

[0003] A characteristic of YwfE is that the L-asparagine at position 108, the L-glutamic acid at position 109, and the L-leucine at position 110 in the amino acid sequence of YwfE are known to be located near the C-terminal amino acid of the dipeptide product (Non-Patent Document 3). Of these, the L-glutamic acid at position 109 contributes to the interaction with magnesium ions, which is important for the enzymatic activity of YwfE (Non-Patent Document 3). The residue corresponding to this L-glutamic acid is widely conserved in L-amino acid α-ligases from various microorganisms (Non-Patent Document 4), suggesting that this L-glutamic acid may be an important amino acid residue for the enzymatic activity of L-amino acid α-ligase.

[0004] Many studies have been conducted on YwfE with modified substrate specificity (Patent Documents 2 to 6, Non-Patent Document 3).

[0005] Patent Document 2 shows that by modifying the amino acid sequence of YwfE at L-phenylalanine at position 62, L-isoleucine at position 92, L-tryptophan at position 332, L-asparagine at position 333, L-methionine at position 334, L-aspartic acid at position 359, and L-aspartic acid at position 361, dipeptides having an acidic amino acid at the N-terminus can be efficiently produced.

[0006] Patent Document 3 shows that carnosine can be efficiently produced by modifying L-isoleucine at position 112 and L-histidine at position 378 in the amino acid sequence of YwfE.

[0007] Patent Document 4 shows that by modifying the amino acid sequence of YwfE at L-asparagine 107, L-aspartic acid 283, L-tryptophan 332, and L-aspartic acid 376, dipeptides having a basic amino acid at the N-terminus can be efficiently produced.

[0008] Patent Document 5 shows that by modifying L-methionine at position 334 in the amino acid sequence of YwfE, a dipeptide having a branched-chain amino acid at the N-terminus is efficiently produced.

[0009] Patent Document 6 shows that imidazole peptides can be efficiently produced by modifying L-asparagine at position 108, L-isoleucine at position 112, and L-histidine at position 378 in the amino acid sequence of YwfE.

[0010] Non-patent document 3 shows that in the L-amino acid ligase TabS of Pseudomonas syringae, substituting serine at position 85, which corresponds to amino acid residue 110 in the amino acid sequence of YwfE, with threonine improves the prolylglycine synthesis activity of TabS.

[0011] On the other hand, there have been no reports to date that modifying a specific amino acid residue in the amino acid sequence of YwfE improves the substrate specificity of L-amino acid α-ligase and reduces the production of by-product dipeptides other than the target dipeptide. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2004 / 058960 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-81404 [Patent Document 3] Japanese Patent Application Laid-Open No. 2013-81405 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-81406 [Patent Document 5] Japanese Patent Application Laid-Open No. 2013-81407 [Patent Document 6] Japanese Patent Application Publication No. 2018-102287 [Non-patent literature]

[0013] [Non-Patent Document 1] Kazuhiko Tabata et al., “ywfE in Bacillus subtilis Codes for a Novel Enzyme, L-Amino Acid Ligase”, J. Bacteriol., 187, p5195-5202 (2005) [Non-patent document 2] Kazuhiko Tabata et al., “Fermentative Production of L-Alanyl-L-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing L-Amino Acid _-Ligase”, Appl. Environ. Microbiol., 73, 20, p6378-6385 (2007) [Non-patent document 3] Yasuhito Shomura, “Structural and enzymatic characterization of BacD, an L-amino acid dipeptide ligase from Bacillus subtilis”, Protein Science, 21, p707-716 (2012) [Non-patent document 4] Haruka Kino, “Effective production of Pro-Gly by mutagenesis of l-amino acid ligase”, J. Biosci. Bioen., 122, 2, p155-159 (2016) Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a method for producing a dipeptide using a protein having dipeptide synthase activity and a microorganism capable of producing said protein, which method reduces the amount of dipeptide by-products other than the target dipeptide by improving the substrate specificity of the protein, and also provides a method for efficiently producing the target dipeptide. [Means for solving the problem]

[0015] The present invention relates to the following 1 to 8. 1. A protein consisting of an amino acid sequence in which one or more amino acid residues selected from the group consisting of positions 107, 108, and 110 in the amino acid sequence represented by SEQ ID NO: 2 have been substituted with the amino acid residues set forth in [1] to [3] below, respectively, and which has L-amino acid α-ligase activity with improved substrate specificity compared to the original protein having the amino acid sequence represented by SEQ ID NO: 2. [1] The 107th amino acid residue is an amino acid residue selected from the group consisting of L-serine, L-alanine, L-histidine, L-glutamine, L-aspartic acid, L-glutamic acid, glycine, and L-methionine. [2] The 108th amino acid residue is an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-proline, L-threonine, L-alanine, L-tyrosine, glycine, and L-leucine. [3] The 110th amino acid residue is an amino acid residue selected from the group consisting of L-methionine, L-valine, L-alanine, L-asparagine, L-cysteine, L-tryptophan, and L-phenylalanine. 2. A protein consisting of an amino acid sequence in which one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence of the original protein described in [4] or [5] below are substituted with amino acid residues described in [1'] to [3'], respectively, and which has L-amino acid α-ligase activity with improved substrate specificity compared to the original protein. [1'] An amino acid residue selected from the group consisting of L-serine, L-alanine, L-histidine, L-glutamine, L-aspartic acid, L-glutamic acid, glycine, and L-methionine for the amino acid residue corresponding to position 107 [2'] For the amino acid residue corresponding to position 108, an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-proline, L-threonine, L-alanine, L-tyrosine, glycine, and L-leucine [3'] An amino acid residue selected from the group consisting of L-methionine, L-valine, L-alanine, L-asparagine, L-cysteine, L-tryptophan, and L-phenylalanine for the amino acid residue corresponding to position 110 [4] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted, and / or added from the amino acid sequence represented by SEQ ID NO: 2, and having L-amino acid α-ligase activity. [5] A homologous protein having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having L-amino acid α-ligase activity. 3. A DNA encoding the protein described in 1 or 2 above. 4. A recombinant DNA containing the DNA described in 3 above. 5. A transformant obtained by transforming a host cell with the recombinant DNA described in 4 above. 6. A method for producing a dipeptide, comprising using the protein described in 1 or 2 above as an enzyme source, causing the enzyme source and two types of L-amino acids to be present in an aqueous medium, producing and accumulating a dipeptide in the aqueous medium, and recovering the dipeptide from the aqueous medium. 7. A method for producing a dipeptide, comprising culturing in a medium a microorganism capable of producing the protein described in 1 or 2 above, producing and accumulating a dipeptide in the culture, and recovering the dipeptide from the culture. 8. The dipeptide is represented by the formula (I) R 1 -R 2 (I) (In the formula, R 1is L-alanine, R 2 and represents an amino acid residue selected from L-glutamine, L-glutamic acid, glycine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, and L-aspartic acid). [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a production method for efficiently producing a specific dipeptide while reducing by-product dipeptides, using a protein having dipeptide synthesis activity with improved substrate specificity and a microorganism capable of producing the protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] 1. Protein of the Present Invention The protein of the present invention is a protein described in (1) or (2) below.

[0018] (1) A protein consisting of an amino acid sequence in which one or more amino acid residues selected from the group consisting of positions 107, 108, and 110 in the amino acid sequence represented by SEQ ID NO: 2 have been substituted with the amino acid residues set forth in [1] to [3] below, respectively, and which has L-amino acid α-ligase activity with improved substrate specificity compared to the original protein having the amino acid sequence represented by SEQ ID NO: 2. [1] The 107th amino acid residue is an amino acid residue selected from the group consisting of L-serine, L-alanine, L-histidine, L-glutamine, L-aspartic acid, L-glutamic acid, glycine, and L-methionine. [2] The 108th amino acid residue is an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-proline, L-threonine, L-alanine, L-tyrosine, glycine, and L-leucine. [3] The 110th amino acid residue is an amino acid residue selected from the group consisting of L-methionine, L-valine, L-alanine, L-asparagine, L-cysteine, L-tryptophan, and L-phenylalanine.

[0019] (2) A protein consisting of an amino acid sequence in which one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence of the parent protein described in [4] or [5] below are substituted with amino acid residues described in [1'] to [3'], respectively, and which has L-amino acid α-ligase activity with improved substrate specificity compared to the parent protein described in [4] or [5]. [1'] An amino acid residue selected from the group consisting of L-serine, L-alanine, L-histidine, L-glutamine, L-aspartic acid, L-glutamic acid, glycine, and L-methionine for the amino acid residue corresponding to position 107 [2'] For the amino acid residue corresponding to position 108, an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-proline, L-threonine, L-alanine, L-tyrosine, glycine, and L-leucine [3'] An amino acid residue selected from the group consisting of L-methionine, L-valine, L-alanine, L-asparagine, L-cysteine, L-tryptophan, and L-phenylalanine for the amino acid residue corresponding to position 110 [4] A mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted and / or added from the amino acid sequence represented by SEQ ID NO: 2, and which has L-amino acid alpha ligase activity. [5] A homologous protein having an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and that has L-amino acid α-ligase activity.

[0020] The base protein described in (2) above is the mutant protein described in [4] or the homologous protein described in [5]. In the amino acid sequence of the base protein, the amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2 refer to the amino acid residues that align at the same positions as the amino acid residues at positions 107, 108, and 110 in the amino acid sequence represented by SEQ ID NO: 2 when the amino acid sequence of the base protein is aligned with the amino acid sequence represented by SEQ ID NO: 2. Hereinafter, the amino acid residues at positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2, as well as the amino acid residues in the amino acid sequence described in [4] or [5] that correspond to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2, are collectively referred to as "amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2."

[0021] In the amino acid sequence of the original protein described in (2) above, the amino acid residues corresponding to the 107th, 108th, and 110th amino acid residues in the amino acid sequence represented by SEQ ID NO: 2 refer to the 107th, 108th, and 110th amino acid residues in the amino acid sequence represented by SEQ ID NO: 3, respectively, when the original protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, which has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2.

[0022] In the amino acid sequence of the protein described in (1) or (2) above, one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2 are substituted with the amino acid residues described in (1)[1] to [3] or (2)[1'] to [3'] above. This can be confirmed by aligning the amino acid sequence of the protein described in (1) or (2) above with the amino acid sequence of the original protein.

[0023] An alignment of amino acid sequences can be created using, for example, the well-known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)]. ClustalW is available, for example, at http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating an alignment using ClustalW, the parameters can be, for example, default values.

[0024] The amino acid residue corresponding to the 107th position in the amino acid sequence represented by SEQ ID NO: 2 is desirably substituted with an amino acid residue selected from the group consisting of L-serine, L-alanine, L-histidine, glycine, and L-methionine, preferably L-alanine, L-histidine, and glycine, among the amino acids [1] or [1'] above, more preferably L-alanine, L-histidine, and glycine.

[0025] The amino acid residue corresponding to the 108th position in the amino acid sequence represented by SEQ ID NO: 2 is desirably substituted with an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-threonine, L-alanine, L-tyrosine, and L-leucine, preferably L-serine, L-threonine, L-alanine, and L-tyrosine, among the amino acids [2] or [2'] above, more preferably L-serine, L-threonine, L-alanine, and L-tyrosine.

[0026] The amino acid residue corresponding to the 110th position in the amino acid sequence represented by SEQ ID NO: 2 is desirably substituted with an amino acid residue selected from the group consisting of L-methionine, L-cysteine, L-tryptophan, and L-phenylalanine, preferably L-methionine, L-tryptophan, and L-phenylalanine, among the amino acids [3] or [3'] above, more preferably L-methionine, L-tryptophan, and L-phenylalanine.

[0027] L-amino acid α-ligase activity refers to the activity of using ATP and two different L-amino acids as substrates to produce a dipeptide in which the two amino acids are peptide-linked at the α-carboxyl groups. L-amino acids here include glycine, which has no stereoisomerism.

[0028] Examples of L-amino acids used as substrates for L-amino acid α-ligase active proteins include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0029] The improved substrate specificity of the protein described in (1) or (2) above compared to the original protein can be confirmed, for example, by the following method. First, a recombinant DNA containing DNA encoding the protein whose activity is to be confirmed is prepared by the method described below. Next, a microorganism lacking L-amino acid α-ligase activity, such as Escherichia coli W3110, is transformed with the recombinant DNA to produce a microorganism, and a cell extract containing the protein is prepared from the resulting culture. The cell extract containing the protein is contacted with an aqueous solution containing the substrate ATP and two L-amino acids to produce a dipeptide in the aqueous solution. Finally, the dipeptide in the reaction solution is detected using HPLC, as described below, and the amounts of the target dipeptide and by-product dipeptide produced are compared to confirm that the substrate specificity is improved compared to the original protein.

[0030] A mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in an original protein, or by artificially inserting or adding amino acid residues into the protein.

[0031] In the mutant protein of [4], the deletion, substitution, insertion or addition of amino acids may mean that 1 to 20 amino acids have been deleted, substituted, inserted or added at any position in the amino acid sequence represented by SEQ ID NO: 2, and for example, 1 to 15, 1 to 10, or 1 to 5 amino acids have been deleted, substituted, inserted or added.

[0032] The amino acids to be substituted, inserted, or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0033] Examples of amino acids that can be substituted for each other are shown below. Amino acids 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 D group: 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

[0034] Homologous proteins are proteins found in living organisms in nature that are a group of proteins that have evolutionary origins derived from the same protein. Homologous proteins are similar in structure and function to each other.

[0035] It is desirable that the amino acid sequence of the homologous protein [5] has at least 80% or more identity to the amino acid sequence represented by SEQ ID NO: 2, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more identity.

[0036] The identity of amino acid sequences or nucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, parameters are, for example, score = 100 and word length = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, parameters are, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0037] A specific example of a homologous protein is the protein of Bacillus amyloliquefaciens represented by SEQ ID NO: 3 (UniprotKB-Q8KWS8).

[0038] The presence of L-amino acid α-ligase activity in the mutant or homologous protein can be confirmed, for example, by the following method. First, recombinant DNA encoding the mutant or homologous protein whose activity is to be confirmed is prepared using the method described below. Next, a microorganism lacking L-amino acid α-ligase activity, such as Escherichia coli W3110, is transformed with the recombinant DNA to produce a microorganism, and a cell extract containing the protein is prepared from the resulting culture. The cell extract containing the protein is contacted with an aqueous solution containing the substrate ATP and two L-amino acids to produce a dipeptide in the aqueous solution. Finally, the presence of L-amino acid α-ligase activity in the mutant or homologous protein can be confirmed by detecting the dipeptide in the reaction solution using HPLC, as described below.

[0039] 2. DNA of the present invention The DNA of the present invention is a DNA that encodes the protein described in (1) or (2) above.

[0040] Specific examples of the DNA of the present invention include DNA encoding a protein having an amino acid sequence in which one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence represented by SEQ ID NO: 2 are substituted with amino acid residues [1] to [3] or [1'] to [3'] above, in the amino acid sequence of a protein encoded by the DNA described in any one of [6] to [8] below. [6] DNA consisting of the base sequence represented by SEQ ID NO: 1 [7] A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a mutant protein or a homologous protein having L-amino acid α-ligase activity. [8] DNA consisting of a nucleotide sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more identity to the nucleotide sequence represented by SEQ ID NO: 1, and encoding a mutant protein or a homologous protein having L-amino acid α-ligase activity.

[0041] In the above, hybridization refers to a process in which DNA hybridizes to a DNA having a specific base sequence or a part of the DNA. Therefore, the base sequence of the DNA having the specific base sequence or the DNA hybridizing to a part of the DNA may be a DNA of a length that is useful as a probe for Northern or Southern blot analysis or can be used as an oligonucleotide primer for PCR analysis.

[0042] DNA used as a probe can be DNA of at least 100 bases or more, preferably 200 bases or more, and more preferably 500 bases or more, and DNA used as a primer can be DNA of at least 10 bases or more, preferably 15 bases or more.

[0043] Methods for DNA hybridization experiments are well known, and hybridization conditions can be determined and experiments can be performed according to numerous standard textbooks, such as Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Methods for General and Molecular Bacteriology (ASM Press, 1994), and Immunology Methods Manual (Academic Press, 1997).

[0044] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with commercially available hybridization kits, such as the Random Primed DNA Labeling Kit (Roche Diagnostics), which uses a random primed probe to prepare probes and hybridize them under stringent conditions.

[0045] The above-mentioned stringent conditions include, for example, incubating a DNA-immobilized filter and probe DNA overnight at 42°C in a solution containing 50% formamide, 5x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2x SSC solution at approximately 65°C.

[0046] The various conditions described above can also be achieved by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to suit the conditions.

[0047] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA consisting of a base sequence that has at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more identity to the base sequence represented by SEQ ID NO: 1, when calculated based on the above parameters using, for example, the above-mentioned programs such as BLAST and FASTA.

[0048] The DNA of the present invention can be obtained, for example, by using DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2 and introducing mutations into a portion of the nucleotide sequence on the DNA encoding one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of the amino acid sequence set forth in SEQ ID NO: 2, using site-directed mutagenesis methods such as those described in Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)) and Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), and substituting the nucleotide sequence with a different amino acid residue. Alternatively, the DNA of the present invention can be obtained using a PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.).

[0049] By a similar method, for example, a mutant protein consisting of an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, and which has L-amino acid alpha ligase activity, can also be obtained by using DNA encoding the mutant protein and, when the amino acid sequence of the mutant protein and the original amino acid sequence represented by SEQ ID NO: 2 are aligned by the method described in 1 above, introducing a mutation into the base sequence of the part of the amino acid sequence of the mutant protein that encodes one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108 and 110 of SEQ ID NO: 2.

[0050] Alternatively, the amino acid sequence of a homologous protein having L-amino acid α-ligase activity and an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:2 can be aligned by the method described above in 1 using DNA encoding the homologous protein. The amino acid sequence of the homologous protein can then be aligned with the amino acid sequence of SEQ ID NO:2, and the amino acid sequence of the homologous protein can be obtained by introducing a mutation into the base sequence of a portion of the amino acid sequence of the homologous protein that encodes one or more amino acid residues selected from the group consisting of amino acid residues corresponding to positions 107, 108, and 110 of SEQ ID NO:2.

[0051] DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 can be obtained, for example, by Southern hybridization of a chromosomal DNA library of a microorganism, preferably a member of the genus Bacillus, more preferably Bacillus subtilis 168 (ATCC23857), using a probe that can be designed based on the nucleotide sequence of DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2, or by PCR [PCR Protocols, Academic Press (1990)] using primer DNAs that can be designed based on the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2 and the chromosomal DNA of Bacillus subtilis 168 (ATCC23857) as a template. Specific examples of DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 2 include DNA having the nucleotide sequence represented by SEQ ID NO: 1.

[0052] A DNA encoding a mutant protein having an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 as described in 1(2)[4] above, and which has L-amino acid alpha ligase activity, can be obtained, for example, by subjecting DNA having the base sequence represented by SEQ ID NO: 1 as a template to error-prone PCR or the like.

[0053] Alternatively, DNA encoding a mutant protein having an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2 in 1(2)[4] above, and having L-amino acid α-ligase activity, can also be obtained by PCR-based site-specific mutagenesis [Gene, 77, 51(1989)], using a pair of PCR primers each having a base sequence at its 5' end designed to introduce the desired mutation (deletion, substitution, insertion or addition).

[0054] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0055] That is, first, a pair of mutagenesis primers is designed with a template of a plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap on the 5' side. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template of a plasmid having the base sequence into which the desired mutation is to be introduced. The amplified fragment obtained in this way can be transformed into Escherichia coli to obtain a plasmid having the base sequence into which the desired mutation has been introduced.

[0056] DNA encoding a homologous protein having an amino acid sequence with 80% or more identity to the amino acid sequence represented by SEQ ID NO:2 and having L-amino acid α-ligase activity as described in 1(2)[5] above can be obtained, for example, by the following method. Specifically, for example, various gene sequence databases are searched for nucleotide sequences with 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity to the nucleotide sequence represented by SEQ ID NO:1, and DNA encoding the homologous protein can be obtained by a method similar to that used to obtain DNA encoding the protein having the amino acid sequence represented by SEQ ID NO:2 above, using probe DNA or primer DNA that can be designed based on the nucleotide sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA.

[0057] The identity of the nucleotide sequence or amino acid sequence can be determined by the same method as in 1 above.

[0058] The DNA of the present invention obtained by the above method can be used as is or cleaved with an appropriate restriction enzyme or the like, and then inserted into a vector by standard methods. The resulting recombinant DNA can then be introduced into host cells, and the base sequence of the DNA can be determined by a commonly used base sequence analysis method, such as the dideoxy method [Proc. Nat. Acad. Sci., USA, 74, 5463 (1977)], or by analysis using a base sequence analyzer such as the Applied Biosystems 3500 Genetic Analyzer or the Applied Biosystems 3730 DNA Analyzer (both manufactured by Thermo Fisher Scientific).

[0059] Examples of vectors that can be used to determine the base sequence of the DNA of the present invention include pBluescriptII KS(+), pPCR-Script Amp SK(+) (both manufactured by Agilent Technologies), pT7Blue (manufactured by Merck Millipore), pCRII (manufactured by Thermo Fisher Scientific), pCR-TRAP (manufactured by Gene Hunter), and pDIRECT [Nucleic Acids Res., 18, 6069 (1990)].

[0060] The host cell may be any cell that can be transformed with the vector and grow in the cell. Examples of the host cell include Escherichia coli DH5α, Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli W3110, Escherichia coli MP347, Escherichia coli NM522, etc.

[0061] Any method for introducing recombinant DNA obtained by incorporating the DNA of the present invention into host cells can be used, as long as it is a method for introducing DNA into host cells. Examples include the method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (JP 63-248394 A), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0062] If the DNA obtained as a result of determining the base sequence is a partial length, full-length DNA can be obtained by Southern hybridization or the like against a chromosomal DNA library using the partial length DNA as a probe.

[0063] Furthermore, the desired DNA can be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on the determined DNA base sequence.

[0064] 3. Recombinant DNA of the Present Invention The recombinant DNA of the present invention is a DNA that can autonomously replicate in a host cell, and is a DNA in which the DNA of the present invention has been incorporated into an expression vector containing a promoter at a position where the DNA of the present invention described above in 2 can be transcribed.

[0065] DNA that can be integrated into a chromosome in a host cell and that contains the DNA of the present invention is also a recombinant DNA of the present invention.

[0066] When the recombinant DNA is capable of being integrated into a chromosome, it does not need to contain a promoter.

[0067] When a prokaryote such as a bacterium is used as a host cell, the recombinant DNA of the present invention is preferably a recombinant DNA comprising a promoter, a ribosome binding sequence, the DNA of the present invention described above in 2, and a transcription termination sequence. It may further comprise a gene that controls the promoter.

[0068] Here, it is preferable to adjust the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon to an appropriate distance, for example, 6 to 18 bases.

[0069] In addition, in the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to place a transcription termination sequence immediately downstream of the structural gene.

[0070] When a microorganism belonging to the genus Escherichia is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of the expression vector include pColdI, pSTV28, pUC118 (all manufactured by Takara Bio Inc.), pET21a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pKYP10 (Japanese Patent Application Laid-Open No. 110600 / 1983), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP-A-63-233798), etc.

[0071] When using the above expression vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of promoters that can be used include promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter.Artificially designed and modified promoters such as a promoter consisting of two trp promoters in tandem, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the letI promoter can also be used.

[0072] When a coryneform bacterium is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Publication No. 58-105999), pCE51, pCE52, and pCE53 [all Molecular and General Genetics, 196, 175 (1984)].

[0073] When using the above expression vector, any promoter that functions in the cells of coryneform bacteria can be used, but for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, p674-679 (2000)] can be used.

[0074] When a yeast strain is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0075] When using the above expression vector, any promoter that functions in the cells of a yeast strain may be used, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0076] The recombinant DNA of the present invention can be produced, for example, by treating the DNA fragment prepared by method 2 above with a restriction enzyme and inserting it downstream of the promoter of the appropriate expression vector described above.

[0077] Here, the expression level of the protein encoded by the DNA of the present invention can be improved by substituting bases in the base sequence of the DNA of the present invention so that the codons are optimal for expression in the host cell. Information on codon usage in host cells is available through public databases.

[0078] 4. Transformant of the Present Invention The transformant of the present invention is a transformant obtained by transforming a host cell with the recombinant DNA of the present invention described in 3 above, i.e., a recombinant DNA containing the DNA of the present invention described in 2 above.

[0079] The host cell into which the recombinant DNA of the present invention is introduced may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm―, Escherichia coli JM109, Escherichia coli HB101, Escherichia coliCJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillusamyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum Prokaryotes such as ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, or Pseudomonas sp.D-0110, or Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia Examples of yeast strains include yeast strains such as Candida pastoris or Candida utilis.

[0080] As the host cell, preferably, a bred strain in which dipeptide decomposition activity has been artificially weakened, a bred strain in which the ability to produce an L-amino acid that is a substrate for the target dipeptide has been artificially imparted or enhanced, or a bred strain that has been subjected to both of these.

[0081] Methods for artificially attenuating the dipeptide decomposition activity of a microorganism used as a host cell include methods for attenuating or blocking at least one enzyme having dipeptide decomposition activity.

[0082] Methods for artificially imparting or enhancing the ability of a microorganism used as a host cell to produce an L-amino acid include (a) a method for alleviating or deactivating at least one of the mechanisms controlling the biosynthetic pathway that produces the target L-amino acid, (b) a method for enhancing the expression of at least one of the enzymes involved in the biosynthetic pathway that produces the target L-amino acid, (c) a method for increasing the copy number of at least one of the enzyme genes involved in the biosynthetic pathway that produces the target L-amino acid, and (d) a method for weakening or blocking at least one of the metabolic pathways that branch off from the biosynthetic pathway that produces the target L-amino acid to a metabolic product other than the target substance. These known methods can be used alone or in combination.

[0083] Specific examples of the above-mentioned methods for attenuating the dipeptide-degrading activity and imparting or enhancing the ability to produce L-amino acids as substrates include known methods such as various genetic engineering methods (Appl Environ Microbiol (2007) 73(20):6378-6385).

[0084] Methods for introducing the recombinant DNA of the above 3 into host cells as an autonomously replicable plasmid include, for example, the above-mentioned calcium ion method, the protoplast method, the electroporation method, the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0085] Methods for integrating recombinant DNA into the chromosome of a host cell include, for example, homologous recombination. Examples of homologous recombination include the use of a homologous recombination plasmid, which can be constructed by ligating a plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. A frequently used homologous recombination method in Escherichia coli is the use of the lambda phage homologous recombination system to introduce recombinant DNA [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0086] Furthermore, E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sensitive to sucrose due to Bacillus subtilis levansucrase incorporated into the chromosome together with recombinant DNA, or a selection method that utilizes the fact that E. coli becomes sensitive to streptomycin when a wild-type rpsL gene is incorporated into E. coli that has a mutant rpsL gene that confers streptomycin resistance [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0087] Whether the transformant obtained by the above method contains the DNA of the present invention described in 2 above can be confirmed, for example, by culturing the transformant in a medium and comparing the amount of the target dipeptide produced and accumulated in the culture with that of the parent strain. Alternatively, it can be confirmed by preparing an extract containing the protein of the present invention from the culture, adding the extract and two different L-amino acids to an aqueous medium, and comparing the amount of the dipeptide produced and accumulated in the aqueous medium with that of the parent strain.

[0088] Examples of such a transformant of the present invention include the transformant described below in the Examples.

[0089] 5. Method for producing the dipeptide of the present invention The method for producing the dipeptide of the present invention is the method described in 5-1 and 5-2 below.

[0090] 5-1. Method for producing dipeptides using L-amino acids as substrates The method for producing the dipeptide of the present invention includes a method for producing a dipeptide using two types of L-amino acids as precursors.

[0091] Specifically, the protein described in (1) or (2) is used as an enzyme source, the enzyme source and two L-amino acids are placed in an aqueous medium, a dipeptide is produced and accumulated in the aqueous medium, and the dipeptide can be collected from the aqueous medium. The enzyme source may be the purified protein described in (1) or (2), or a culture obtained by culturing the transformant described in 4 above, i.e., a microorganism capable of producing the protein described in (1) or (2), in a medium, or a processed product of the culture. When the purified protein described in (1) or (2) is used as the enzyme source, ATP is placed in an aqueous solvent together with the two L-amino acids. Here, the culture includes a cultured microorganism capable of producing the protein described in (1) or (2) and a medium, and the culture contains the protein described in (1) or (2) expressed by the microorganism.

[0092] The origin of the ATP and the two L-amino acids does not matter as long as they are substrates for the protein of the present invention possessed by the transformant of the present invention. A culture of a microorganism capable of producing ATP and the two L-amino acids or a processed product of the culture may be used as is, or ATP and the two L-amino acids extracted from the culture or a processed product of the culture may be used.

[0093] The culture or a processed product of the culture contains the protein described in (1) or (2) and ATP as an enzyme source. Examples of the processed product of the culture include a concentrate of the culture, a dried product of the culture, bacterial cells obtained by centrifuging the culture, a dried product of the bacterial cells, a freeze-dried product of the bacterial cells, a surfactant-treated product of the bacterial cells, an ultrasonically-treated product of the bacterial cells, a mechanically ground product of the bacterial cells, a solvent-treated product of the bacterial cells, an enzyme-treated product of the bacterial cells, a protein fraction of the bacterial cells, an immobilized product of the bacterial cells, and an enzyme preparation obtained by extraction from the bacterial cells.

[0094] Examples of aqueous media include water, phosphates, carbonates, acetates, borates, citrates, buffer solutions such as Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. The culture medium of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0095] The dipeptide produced in the aqueous medium can be analyzed by a conventional method using high performance liquid chromatography, etc. The dipeptide can be collected from the aqueous medium by a conventional method using activated carbon, an ion exchange resin, etc.

[0096] 5-2. Fermentation method for producing dipeptides The method for producing a dipeptide of the present invention includes a fermentation method for producing a dipeptide, which comprises culturing the transformant described in 4 above, i.e., a microorganism capable of producing the protein described in (1) or (2) above, in a medium, producing and accumulating the dipeptide in the culture, and recovering the dipeptide from the culture. Here, the culture includes a cultured microorganism capable of producing the protein described in (1) or (2) above and a medium, and the culture contains the protein described in (1) or (2) above expressed by the microorganism.

[0097] The transformant of the present invention used in the method for producing a dipeptide by fermentation is preferably a transformant capable of producing an L-amino acid that serves as a substrate for the dipeptide.

[0098] The transformant in item 4 above can be cultured according to a conventional method used for culturing microorganisms.

[0099] The medium for culturing the transformant may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the transformant and allows the transformant to be cultured efficiently.

[0100] The carbon source may be any that can be utilized by the transformant, and examples thereof include sugars such as glucose, fructose, sucrose, molasses containing these, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as glycerol, ethanol, and propanol.

[0101] Examples of nitrogen sources that can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.

[0102] Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0103] In the method for producing a dipeptide by fermentation, if the transformant used does not have the ability to produce the L-amino acid that serves as the substrate, the L-amino acid is added to the medium during cultivation.

[0104] Furthermore, in a method for producing a dipeptide by fermentation, if the transformant used does not have the ability to produce the L-amino acid substrate, instead of adding the L-amino acid to the medium during cultivation, the L-amino acid may be supplied to the transformant of the present invention by co-culturing a microorganism capable of producing the L-amino acid from sugar with the transformant of the present invention.

[0105] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH of the culture solution during cultivation is usually maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0106] Furthermore, antibiotics such as ampicillin or tetracycline may be added to the medium as needed during culture. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using the trp promoter.

[0107] By the above-mentioned cultivation, dipeptides are produced and accumulated in the culture, and the dipeptides can be produced by collecting them from the culture.

[0108] The obtained dipeptides can be analyzed by a conventional method using high performance liquid chromatography, etc. Dipeptides can be collected from the above culture or a treated product of the culture by a conventional method using activated carbon, ion exchange resin, etc. When dipeptides accumulate within the cells, for example, the cells can be disrupted by ultrasound, etc., and the cells can be removed by centrifugation, and the dipeptides can be collected from the resulting supernatant using activated carbon, ion exchange resin, etc.

[0109] The dipeptide produced by the production method of the present invention is represented by the formula (I) R 1 -R 2 (I) (In the formula, R 1 is L-alanine, R 2 represents an amino acid residue selected from L-glutamine, L-glutamic acid, glycine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, and L-aspartic acid) It is a dipeptide represented by the formula: [Example]

[0110] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0111] [Analysis example] In the examples, L-alanyl-L-glutamine and L-alanyl-L-alanine were analyzed and quantified by HPLC (Shimadzu Corporation) according to the following procedures. Analysis conditions Column: InertSustain 3 μm 3 mm × 150 mm (GL Sciences) Column temperature: 40℃ Mobile phase: 4.08 g / L potassium phosphate, 6.07 g / L sodium heptanesulfonate, and 125 mL / L acetonitrile (pH 2.5, adjusted with phosphoric acid) Flow rate: 0.4mL / min Detection wavelength: 210 nm

[0112] [Example 1] Construction of a microorganism expressing mutant YwfE (1) Preparation of an expression vector for L-amino acid α-ligase (YwfE) from Bacillus subtilis 168 strain PCR was performed using the genomic DNA of Bacillus subtilis 168 strain prepared by a standard method as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 4 and 5 as a primer set, to obtain a DNA fragment of approximately 1.4 kbp containing the ywfE gene.

[0113] The ywfE fragment obtained above was ligated to the expression vector pET-21a(+) (Merck Millipore) using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pET21a-ywfE.

[0114] (2) Construction of microorganisms expressing mutant YwfE Using the plasmid pET21a-ywfE obtained in (1) above as a template and the PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.), a total of 57 types of plasmids were constructed in which the amino acid residue at position 107, 108, or 110 in the amino acid sequence of YwfE was replaced with another amino acid, using DNA consisting of the base sequence represented by the "primer set" listed in any of Tables 1 to 3 as a primer set.

[0115] When substituting L-Asn at position 107 in the amino acid sequence of YwfE, the primer set listed in Table 1 was used; when substituting L-Asn at position 108, the primer set listed in Table 2 was used; and when substituting L-Leu at position 110, the primer set listed in Table 3 was used. [Table 1] [Table 2] [Table 3]

[0116] The wild-type YwfE expression plasmid pET21a-ywfE obtained in (1) above and a total of 57 mutant YwfE expression plasmids containing the above mutation sites were used to transform BL21(DE3)pLysS (Merck Millipore) to obtain transformants containing each plasmid.

[0117] [Example 2] Dipeptide production using mutant YwfE A total of 58 transformants obtained in Example 1 above were cultured on LB plates at 30°C for 24 hours, then inoculated into large test tubes containing 5 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 20 hours. Subsequently, 0.1 mL of the resulting culture was inoculated into a flask containing 50 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C. After the bacterial cell OD600 reached a range of 0.4 to 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was continued with shaking for an additional 6 hours. After the culture was completed, the culture was centrifuged to collect the bacterial cells.

[0118] A crude protein extract was obtained from the resulting bacterial cells by standard methods, and wild-type YwfE or each mutant YwfE enzyme was recovered from the extract using a His Buffer Kit (GE Healthcare). The recovered solution was diluted with Buffer A [50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10% glycerol] and concentrated and desalted using an Amicon Ultra-4 (Merck Millipore) to obtain each purified YwfE enzyme.

[0119] Using the obtained wild-type YwfE purified enzyme and mutant YwfE purified enzyme, L-alanyl-L-glutamine (hereinafter referred to as L-Ala-L-Gln) was produced by the following method, and the effects of each mutation were compared.

[0120] (1) Dipeptide production using mutant YwfE with L-Asn substitution at position 107 The production of L-Ala-L-Gln by enzymatic reaction was investigated using a total of 19 purified enzymes, including the wild-type YwfE obtained above and purified mutant YwfE enzymes in which the L-Asn at position 107 in the amino acid sequence of YwfE was replaced with the amino acid residue listed in Table 1 above.

[0121] A 0.1 mL reaction mixture consisting of 22.5 μg of each purified enzyme, 100 mM Tris-HCl (pH 9.5), 30 mM magnesium sulfate, 30 mM disodium ATP, 40 mM L-Ala, and 40 mM L-Gln was prepared and subjected to dipeptide synthesis at 37°C for 1 hour. After the reaction, the dipeptides produced were analyzed by HPLC. The amounts (mM) of L-Ala-L-Gln and L-alanyl-L-alanine (hereafter referred to as L-Ala-L-Ala) produced by the enzymatic reaction with each purified enzyme, as well as the enzymatic activity of each purified enzyme (L-Ala-L-Gln synthesis activity ratio and L-Ala-L-Ala ratio), are shown in Table 4.

[0122] In Table 4, "L-Ala-L-Gln synthesis activity ratio" represents the value for each mutant YwfE obtained by dividing the amount of L-Ala-L-Gln produced per unit time by the purified enzyme concentration (mM) in the reaction solution, with the value for wild-type YwfE set to 1.00. In Table 4, "L-Ala-L-Ala ratio" represents the value for each mutant YwfE obtained by dividing the amount of L-Ala-L-Ala produced (mM) by the amount of L-Ala-L-Gln produced (mM), with the value for wild-type YwfE set to 1.0. [Table 4]

[0123] As a result, as shown in Table 4, mutant YwfE in which L-Asn at position 107 in the amino acid sequence of YwfE was substituted with L-Ser, L-Ala, L-His, L-Gln, L-Asp, L-Glu, Gly, or L-Met had a reduced production ratio of L-Ala-L-Ala compared to wild-type YwfE. Among these, mutant YwfE in which L-Asn at position 107 in the amino acid sequence of YwfE was substituted with L-Ser, L-Ala, L-His, Gly, or L-Met also had an improved production amount of L-Ala-L-Gln compared to wild-type YwfE.

[0124] From the above, it was found that by using mutant YwfE in which L-Asn at position 107 in the amino acid sequence of YwfE is replaced with L-Ser, L-Ala, L-His, Gly, or L-Met, the production ratio of the by-product dipeptide L-Ala-L-Ala is reduced and the production amount of L-Ala-L-Gln is improved compared to wild-type YwfE.

[0125] (2) Dipeptide production using mutant YwfE with L-Asn substitution at position 108 The production of L-Ala-L-Gln by enzymatic reaction was investigated using a total of 19 purified enzymes, including the wild-type YwfE obtained above and purified enzymes of mutant YwfE in which L-Asn at position 108 in the amino acid sequence of YwfE was replaced with the amino acid residue listed in Table 2 above.

[0126] A 0.1 mL reaction mixture containing 15 μg of purified enzyme (13.6 μg for the mutant YwfE enzyme substituted with L-Phe), 100 mM Tris-HCl (pH 9.5), 30 mM magnesium sulfate, 30 mM disodium ATP, 40 mM L-Ala, and 40 mM L-Gln was prepared and subjected to dipeptide synthesis at 37°C for 1 hour. The dipeptides were analyzed by HPLC. Table 5 shows the 108th amino acid residue of YwfE used in each reaction, the amounts (mM) of L-Ala-L-Gln and L-Ala-L-Ala produced, and the enzyme activity. The "L-Ala-L-Gln synthesis activity ratio" and "L-Ala-L-Ala ratio" in Table 5 are the same as those in (1) above. [Table 5]

[0127] As a result, mutant YwfE in which L-Asn at position 108 of the amino acid sequence of YwfE was substituted with L-Phe, L-Met, L-Ser, L-Pro, L-Thr, L-Ala, L-Tyr, Gly, or L-Leu showed a reduced production ratio of L-Ala-L-Ala compared to wild-type YwfE. Among these, mutant YwfE in which L-Asn at position 108 of the amino acid sequence of YwfE was substituted with L-Phe, L-Met, L-Ser, L-Thr, L-Ala, L-Tyr, or L-Leu also showed an improved production of L-Ala-L-Gln compared to wild-type YwfE.

[0128] From the above, it was found that by using mutant YwfE in which L-Asn at position 108 in the amino acid sequence of YwfE is replaced with L-Phe, L-Met, L-Ser, L-Thr, L-Ala, L-Tyr, or L-Leu, the production ratio of the by-product dipeptide L-Ala-L-Ala is reduced and the production amount of L-Ala-L-Gln is improved compared to wild-type YwfE.

[0129] (3) Dipeptide production using mutant YwfE with L-Leu substitution at position 110 The production of L-Ala-L-Gln by enzymatic reaction was investigated using a total of 19 purified enzymes, including the wild-type YwfE obtained above and purified mutant YwfE enzymes in which the L-Leu at position 110 in the amino acid sequence of YwfE was replaced with the amino acid residue listed in Table 3 above.

[0130] A 0.1 mL reaction mixture containing 22.5 μg of purified enzyme, 100 mM Tris-HCl (pH 9.5), 30 mM magnesium sulfate, 30 mM disodium ATP, 40 mM L-Ala, and 40 mM L-Gln was prepared and subjected to dipeptide synthesis at 37°C for 1 hour. The dipeptides were analyzed by HPLC. Table 6 shows the 110th amino acid residue of YwfE used in each reaction, the amounts (mM) of L-Ala-L-Gln and L-Ala-L-Ala produced, and the enzyme activity. The "L-Ala-L-Gln synthesis activity ratio" and "L-Ala-L-Ala ratio" in Table 6 are the same as those in (1) above. [Table 6]

[0131] As a result, mutant YwfE in which L-Leu at position 110 in the amino acid sequence of YwfE was substituted with L-Met, L-Val, L-Ala, L-Asn, L-Cys, L-Trp, or L-Phe showed a reduced production ratio of L-Ala-L-Ala compared with wild-type YwfE. Among these, mutant YwfE in which L-Leu at position 110 in the amino acid sequence of YwfE was substituted with L-Met, L-Cys, L-Trp, or L-Phe also showed an improved production of L-Ala-L-Gln compared with wild-type YwfE.

[0132] From the above, it was found that by using mutant YwfE in which L-Leu at position 110 in the amino acid sequence of YwfE is replaced with L-Met, L-Cys, L-Trp, or L-Phe, the production ratio of the by-product dipeptide L-Ala-L-Ala is reduced and the production amount of L-Ala-L-Gln is improved compared to wild-type YwfE.

[0133] [Example 3] Production of dipeptides by fermentation using microorganisms carrying mutant YwfE (1) Creation of microorganisms Using the plasmid pPE167 described in Non-Patent Document 2, which contains wild-type YwfE, as a template and the PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.), a total of 10 types of mutant YwfE expression plasmids were constructed, in which either the 107th, 108th, or 110th amino acid in the amino acid sequence of YwfE was replaced with another amino acid, using DNA consisting of the base sequences shown in the "primer set" in Table 7 as a primer set.

[0134] The wild-type YwfE expression plasmid pPE167 and the above 10 mutant YwfE expression plasmids were used to transform the JKYPQ3 strain described in Non-Patent Document 2, to obtain transformants having each plasmid. [Table 7]

[0135] (2) Production of dipeptides The resulting transformants were cultured on LB plates containing 100 mg / L kanamycin at 30°C for 24 hours, and then inoculated into a wide test tube containing 5 mL of LB medium containing 10 g / L glucose and 100 mg / L kanamycin, followed by shaking culture at 30°C for 20 hours. Subsequently, 0.1 mL of the resulting culture broth was inoculated into a wide test tube containing 5 mL of production medium containing 100 mg / L kanamycin [glucose 30 g / L, magnesium sulfate heptahydrate 1 g / L, casamino acids 5 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 16 g / L, potassium dihydrogen phosphate 14 g / L, trisodium citrate dihydrate 1 g / L, L-proline 0.4 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, and manganese sulfate pentahydrate 10 mg / L (all components except glucose and magnesium sulfate heptahydrate were adjusted to pH 7.2 with aqueous sodium hydroxide solution and then autoclaved; aqueous solutions containing glucose and magnesium sulfate heptahydrate were prepared separately, autoclaved, cooled, and mixed)], and the resulting medium was cultured with shaking at 30°C for 48 hours.

[0136] After the cultivation was completed, the culture medium was centrifuged, and the concentrations (g / L) of L-Ala-L-Gln and L-Ala-L-Ala in the supernatant were analyzed by HPLC. The results are shown in Table 8. In Table 8, "L-Ala-L-Ala / L-Ala-L-Gln" represents the value (%) obtained by dividing the amount of L-Ala-L-Ala produced (g / L) by the amount of L-Ala-L-Gln produced (g / L). [Table 8]

[0137] As a result, it was found that by using a microorganism capable of expressing mutant YwfE in which the L-Asn at position 107 of YwfE was replaced with L-Ala, L-His, or Gly, the L-Asn at position 108 with L-Ser, L-Thr, L-Ala, or L-Tyr, and the L-Leu at position 110 with L-Met, L-Trp, or L-Phe, the production amount of L-Ala-L-Gln was improved and the ratio of by-product L-Ala-L-Ala to L-Ala-L-Gln was reduced compared to a microorganism expressing wild-type YwfE, and that the above mutations improved the substrate specificity of YwfE. [Industrial Applicability]

[0138] The present invention provides a protein having dipeptide synthesis activity with improved substrate specificity, and a method for efficiently producing a target dipeptide while reducing by-product dipeptides other than the target dipeptide, using the protein or a microorganism capable of producing the protein.

Claims

1. A protein consisting of an amino acid sequence in which the 107th, 108th, or 110th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 has been substituted with an amino acid residue described in [1] to [3] below, respectively, and which has L-amino acid α-ligase activity with improved substrate specificity compared to the original protein having the amino acid sequence represented by SEQ ID NO:

2. [1] For the 107th amino acid residue, an amino acid residue selected from the group consisting of L-alanine, L-histidine, L-glutamine, L-aspartic acid, L-glutamic acid, glycine, and L-methionine [2] For the 108th amino acid residue, an amino acid residue selected from the group consisting of L-phenylalanine, L-methionine, L-serine, L-proline, L-threonine, L-tyrosine, glycine, and L-leucine. [3] For the 110th amino acid residue, an amino acid residue selected from the group consisting of L-methionine, L-valine, L-asparagine, L-cysteine, L-tryptophan, and L-phenylalanine.

2. A DNA encoding the protein of claim 1.

3. A recombinant DNA comprising the DNA of claim 2.

4. A transformant obtained by transforming a host cell with the recombinant DNA according to claim 3.

5. A method for producing a dipeptide, comprising using the protein according to claim 1 as an enzyme source, causing the enzyme source and two kinds of L-amino acids to be present in an aqueous medium, producing and accumulating a dipeptide in the aqueous medium, and collecting the dipeptide from the aqueous medium.

6. A method for producing a dipeptide, comprising culturing a microorganism capable of producing the protein of claim 1 in a medium, producing and accumulating the dipeptide in the culture, and recovering the dipeptide from the culture.

7. The dipeptide is of formula (I) R １ -R ２ (I) (In the formula, R １ is L-alanine, R ２ represents an amino acid residue selected from L-glutamine, L-glutamic acid, glycine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, and L-aspartic acid.

7. The method according to claim 5 or 6, wherein the dipeptide is represented by the formula:

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