JP2012044909A - Production of amide and peptide by wild type 4-coumaroyl coa ligase and mutated enzyme thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means in which an amide bond forming method, which neither cause racemization nor need a protective group, is made possible by applying a modified biocatalytic function to amide bond formation which has previously depended on organic synthesis.SOLUTION: The mutated enzyme of 4-Coumaroyl CoA ligase has at least one amino acid substitution in the amino acid sequence forming the carboxylic acid binding pocket of 4-coumaroyl CoA ligase. The mutated enzyme gains the ability to synthesize at least dipeptide by the substitution. To be concrete, the substitution is from a tyrosine residue to a phenylalanine residue or from a glutamine residue to an alanine residue.

Description

  The present invention relates to a novel plant-derived secondary metabolic enzyme and a novel use of the enzyme.

Some of the secondary metabolic enzymes involved in the biosynthesis of natural organic compounds have extremely tolerant substrate specificity and potential catalytic ability, and the enzyme catalytic function changes greatly due to subtle differences in structure. Can be a good model for functional control and creation of molecular diversity.
4 Coumaroyl CoA synthase (4-Coumaroyl-CoA Ligase; 4CL) is a key enzyme in the biosynthesis of plant phenylpropanoids such as flavonoids and lignins. 4CL is an enzyme that produces 4-coumaroyl CoA thioester using 4-coumaric acid, ATP, and CoASH as substrates. This enzyme reaction consists of two steps. First, 4-coumaric acid is activated by being converted to an adenylation intermediate by ATP, and then the reaction is terminated by forming a thioester with CoASH (FIG. 1, non-patented). Reference 1).

  Among 4CL derived from Arabidopsis thaliana, At4CL2 is a soluble protein composed of a monomer having a molecular weight of 60 kDa that can be heterologously expressed in E. coli, and is a relatively stable and well-studied enzyme (non-patent document). 2, 3). Based on a homology model based on the crystal structure of the adenylation domain of gramicidin S synthase (NRPS) as a template, site-specific mutations have been introduced into the initiation substrate carboxylic acid binding pocket, thereby changing the specificity for the initiation substrate. However, although it has been reported that cinnamic acid and sinapinic acid are activated instead of coumaric acid (Non-patent Document 3), there is no report on the specificity of the thiol side substrate.

  In this way, the process of activating the carboxyl group of the starting substrate to the adenylation intermediate includes fatty acid CoA synthase, non-ribosome-dependent peptide synthase (NRPS) adenylation domain, luciferase, etc. (Figure 1). In fact, these series of enzymes conserve the sequence of the AMP binding site and constitute the ANL (Acyl-CoA synthetases, the NPRS adenylation domains, the Luciferase enzymes) superfamily enzymes. It is known (Non-Patent Document 1). Although the ANL superfamily enzymes generally exhibit low amino acid sequence homology to each other, they have a similar overall protein structure composed of a large N-terminal domain to which carboxylic acids bind and a small C-terminal domain containing catalytic residues. It has been reported that the C-terminal domain undergoes a large dynamic structural change as the adenylation reaction proceeds (Non-patent Document 1). Interestingly, recently, an acyl CoA synthase derived from the nitrile-degrading bacterium Pseudomonas chlororaphis has formed an amide C—N bond instead of the original thioester CS bond using L-cysteine as a substrate in addition to CoA. It has been reported to produce N-acylcysteine (Non-patent Document 4).

  Peptide compounds composed of amides and amide bonds have various physiological activities as analogs of in vivo components such as proteins, and are expected from the field of pharmaceutical development as peptide drug discovery. Conventionally, the synthesis of peptides has exclusively used organic chemical techniques. In the organic chemical synthesis of peptides, an amide bond is formed using an active ester such as succinimide, a condensing agent such as dicyclohexylcarbodiimide and WSCI, and an activator such as HOBt. However, the chemical synthesis method has problems such as easily causing racemization and protecting functional groups not involved in condensation in advance. If there is a method for overcoming these problems and forming an amide bond that does not require racemization and does not require unnecessary protecting groups, its utility value is extremely high.

  On the other hand, an enzyme that synthesizes a (poly) amide derived from a specific amino acid is known, and the above-mentioned acyl CoA synthase derived from the nitrile-degrading bacterium Pseudomonas chlororaphis also forms an amide bond with L-cysteine. However, enzymes that activate the carboxyl group of the starting substrate and catalyze the thioester bond can be found in fatty acid CoA synthase, NRPS, in addition to 4CL. There are no research examples.

Gulick, A. M., ACS Chem. Biol. 4, 811-827 (2009) Stuible, H.-P. and Kombrink, E., J. Biol. Chem. 276, 26893-26897 (2001) Schneider, K. et al., Proc. Natl. Acad. Sci. USA 100, 8601-8606 (2003) Abe, T. et al., J. Biol. Chem. 283, 11312-11321 (2008)

  As described above, an enzyme that recognizes various amino acids as a substrate and condenses from a dipeptide to a polyamide (polypeptide) is not known. The problem to be solved by the present invention is that, by applying a modified biocatalytic function to the formation of an amide bond that has conventionally relied on organic synthesis, an amide bond that does not cause racemization and does not require a protecting group. The object is to provide a means and the like capable of forming.

  The present inventors paid attention to 4 coumaroyl CoA synthase that catalyzes the formation of a thioester bond of coumaric acid, and found that the enzyme generates not only a thioester bond but also an amide bond. The present inventors further intensively studied the three-dimensional structure of the enzyme and created a single amino acid substitution mutant of the wild-type enzyme. It was verified that the created mutant enzyme overcomes the substrate specificity barrier specific to the wild type, and has acquired a highly versatile peptide synthesis ability, thereby completing the present invention.

That is, the present invention is as follows.
[1] A mutant type 4 coumaroyl CoA synthase in which at least one amino acid in the amino acid sequence constituting the carboxylic acid binding pocket of 4 coumaroyl CoA synthase is substituted, wherein the enzyme has at least dipeptide synthesis ability by the substitution. .
[2] The mutant 4 coumaroyl CoA synthetase according to [1], wherein the substitution is a substitution from a tyrosine residue to a phenylalanine residue or a substitution from a glutamine residue to an alanine residue.
[3] The mutant 4 coumaroyl CoA synthetase according to [1] or [2], which comprises the following amino acid sequences (A1) to (A10):
(A1) an amino acid sequence in which the tyrosine residue at position 253 is substituted with a phenylalanine residue or the glutamine residue at position 345 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 1;
(A2) an amino acid sequence in which the tyrosine residue at position 260 is substituted with a phenylalanine residue or the glutamine residue at position 352 is replaced with an alanine residue in the amino acid sequence of SEQ ID NO: 2;
(A3) an amino acid sequence in which the tyrosine residue at position 263 is substituted with a phenylalanine residue or the glutamine residue at position 355 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 3;
(A4) an amino acid sequence in which the tyrosine residue at position 268 is substituted with a phenylalanine residue or the glutamine residue at position 360 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 4;
(A5) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 5 is substituted with a phenylalanine residue or the glutamine residue at position 333 is substituted with an alanine residue;
(A6) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 6 is substituted with a phenylalanine residue, or the glutamine residue at position 333 is substituted with an alanine residue;
(A7) an amino acid sequence in which the tyrosine residue at position 246 is substituted with a phenylalanine residue in the amino acid sequence of SEQ ID NO: 7 or the glutamine residue at position 338 is substituted with an alanine residue;
(A8) an amino acid sequence in which the tyrosine residue at position 239 is substituted with a phenylalanine residue or the glutamine residue at position 331 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 8;
(A9) the amino acid sequence of SEQ ID NO: 9 or 10; or (A10) at a position other than the substitution site of any one of the amino acid sequences of (A1) to (A9), further one to several amino acids are substituted or missing. An amino acid sequence into which a loss or addition mutation has been introduced, wherein the protein comprising the amino acid sequence after the mutation is capable of synthesizing a dipeptide.
[4] A gene encoding the mutant 4 coumaroyl CoA synthetase according to any one of [1] to [3].
[5] The gene according to [4], comprising the following polynucleotides (B1) to (B10):
(B1) In the base sequence of SEQ ID NO: 11, the base sequence encoding tyrosine at positions 757 to 759 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1033 to 1035 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B2) In the base sequence of SEQ ID NO: 12, the base sequence encoding tyrosine at positions 778 to 780 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1054 to 1056 is a base encoding alanine A polynucleotide comprising a base sequence substituted with a sequence;
(B3) In the base sequence of SEQ ID NO: 13, the base sequence encoding tyrosine at positions 787 to 789 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1063 to 1065 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B4) In the base sequence of SEQ ID NO: 14, the base sequence encoding tyrosine at positions 802 to 804 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1078 to 1080 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B5) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 15 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B6) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 16 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B7) In the base sequence of SEQ ID NO: 17, the base sequence encoding tyrosine at positions 736 to 738 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1012 to 1014 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B8) In the base sequence of SEQ ID NO: 18, the base sequence encoding tyrosine at positions 715 to 717 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 991 to 993 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B9) a polynucleotide comprising the base sequence of SEQ ID NO: 19 or 20, or (B10) a polynucleotide comprising a base sequence having 70% or more sequence identity to any of the base sequences (B1) to (B9) above A polynucleotide encoding an enzyme having the ability to synthesize dipeptides.
[6] A recombinant vector containing the gene according to [4] or [5].
[7] A transformant into which the recombinant vector according to [6] has been introduced.
[8] A compound having an amide bond, comprising the step of reacting the mutant 4 coumaroyl CoA synthetase according to any one of [1] to [3] in the presence of a substrate containing a carboxylic acid, a substrate containing an amine, and ATP. Manufacturing method.
[9] A method for producing a compound having an amide bond, comprising a step of culturing the transformant according to [7] in the presence of a substrate containing a carboxylic acid, a substrate containing an amine, and ATP.
[10] The production method according to [8] or [9], wherein the substrate containing carboxylic acid and the substrate containing amine are amino acids, and the compound having an amide bond is a peptide.
[11] A method for producing a compound having an amide bond, comprising a step of reacting 4 coumaroyl CoA synthase in the presence of coumaric acid or other carboxylic acid, an amine-containing substrate and ATP in the absence of CoASH.
[12] The production method according to [11], wherein the compound having an amide bond is coumaric acid amide.
[13] The production method according to [11], wherein the compound having an amide bond is capsaicin.
[14] The production method according to any one of [11] to [13], wherein the 4 coumaroyl CoA synthase is a wild type 4 coumaroyl CoA synthase.

  The mutant type 4 coumaroyl CoA synthetase of the present invention is a highly versatile enzyme with greatly expanded substrate specificity compared to the wild type enzyme, and can form amide bonds between various substrates. The method for producing an amide compound using the mutant 4 coumaroyl CoA synthetase of the present invention can efficiently produce dipeptides, bioactive amides and the like under clean and mild conditions as compared with the organic synthesis method. The mutant 4 coumaroyl CoA synthetase and the production method using the same of the present invention can be used as various raw materials for pharmaceuticals, cosmetics, and foods (functional foods) by selecting appropriate carboxylic acids and amines as substrates. Amide compounds can be provided.

FIG. 1 shows a comparison of the reaction mechanism of 4CL and ANL superfamily enzymes. FIG. 2 shows amide ligase activity for various L-amino acids of 4CL derived from A. thaliana. The vertical axis of the graph represents the total amount (μM) of AMP calculated from the peak ratio of AMP in the obtained HPLC result. FIG. 3 shows the chemical structure of coumaric acid amide produced by A.thaliana-derived 4CL. Figure 4 shows the chemical structure of a bioactive amide produced by the tolerant substrate specificity exhibited by 4CL from A. thaliana. Fig. 5 shows a homology model (4D whole structure: left) of A.thaliana-derived 4CL and a coumaric acid binding site (right) inferred from the model. FIG. 6 shows the CoA synthesis activity (left) and dipeptide synthesis activity (right) of 4CL wild type and mutant enzymes. In the figure, WT indicates wild type. The vertical axis of the graph represents the total amount (μM) of AMP calculated from the peak ratio of AMP in the obtained HPLC result. FIG. 7 shows dipeptide synthesis activity for various L-amino acids of 4CL Q345A mutant enzyme. The vertical axis of the graph represents the total amount (μM) of AMP calculated from the peak ratio of AMP in the obtained HPLC result. FIG. 8 shows the chemical structure of the 4CL transition state intermediate analog.

  Hereinafter, in the present specification, indications using abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are defined in IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 ( 1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

1. Mutant 4 Coumaroyl CoA synthase (mutant 4CL)
The present invention relates to a mutant 4 coumaroyl CoA synthase (hereinafter referred to as “mutant 4CL of the present invention” or “mutant 4CL” in which at least one amino acid in the amino acid sequence constituting the carboxylic acid binding pocket of 4 coumaroyl CoA synthase is substituted. May be abbreviated as “).

  4 Coumaroyl CoA synthase (hereinafter sometimes abbreviated as “4CL”) is also called 4-coumarate CoA ligase, and is an enzyme that produces 4 coumaroyl CoA, which is a substrate for phenylpropanoid pathway biosynthesis. EC. It is an enzyme belonging to 6.2.1.12. “Mutant 4CL” is included in “4CL” when classified from the viewpoint that it retains the activity of producing 4 coumaroyl CoA. “Wild type 4CL” is used for the purpose of excluding “mutant 4CL” from “4CL”.

  The 4CL carboxylic acid binding pocket refers to a binding site with a 4CL carboxylic acid substrate newly constructed by the present inventors based on a homology model using luciferase as a template. FIG. 5 shows a homology model for At4CL2 derived from Arabidopsis thaliana and a binding pocket with the substrate coumaric acid.

  In the case of At4CL2 (SEQ ID NO: 1), the amino acid residues constituting the carboxylic acid binding pocket are Ile-252, Tyr-253, Asn-256, Met-293, Lys-320, Gly-322, Ala-323, Gly -346, Gly-348, Pro-354, Val-355 and Leu-356 (Schneider, K. et al., Proc. Natl. Acad. Sci. USA 100, 8601-8606 (2003), Fig. 3 See). In the present invention, based on the homology between the amino acid sequence of the carboxylic acid binding pocket found from At4CL2 and the amino acid sequence of the other 4CL, it exists in the carboxylic acid binding pocket of other 4CL other than At4CL2 and the pocket. It is possible to analogize the position of the corresponding amino acid residue.

  The substitution of at least one amino acid in the amino acid sequence constituting the carboxylic acid binding pocket is a substitution for obtaining a dipeptide synthesis ability not found in the wild type enzyme. Preferably, substitution from tyrosine residue to phenylalanine residue or substitution from glutamine residue to alanine residue in the amino acid sequence constituting the carboxylic acid binding pocket, substitution from tyrosine residue to phenylalanine residue and glutamine It may be a 2-amino acid substitution consisting of a substitution of a residue to an alanine residue.

  The 1 amino acid substitution described above can be introduced into a gene encoding 4CL by a method known per se such as site-directed mutagenesis.

  “At least dipeptide synthesis ability” in the present invention “obtain at least dipeptide synthesis ability” refers to the property of synthesizing a homodipeptide in the presence of ATP using any amino acid (one kind) as a substrate. The ability to synthesize dipeptides may be a property of synthesizing a dipeptide consisting of two amino acids, or a property of synthesizing a polypeptide consisting of one or more amino acids. “Dipeptide synthesis ability” is measured by reacting the enzyme of the present invention with any amino acid (one species) as a substrate in the presence of ATP, and the reaction product is subjected to the presence or absence of dipeptide synthesis using chromatography such as TLC or HPLC. This can be done by detecting. Details of the measurement method are described in Example 8.

  The 4CL used in the present invention may be 4CL derived from any plant or bacteria. For example, Arabidopsis thaliana, Eucalyptus camaldulensis, Populus trichocarpa, Nicotiana tabacum, Galega orientalis, Pinus massoniana, Pinus taeda, etc. However, it is not limited to these.

  Wild type 4CL is derived from Arabidopsis thaliana 4CL2 (AT3G21240, GeneID: 821678, SEQ ID NO: 1), 4CL1 (AT1G51680, GeneID: 841593, SEQ ID NO: 2), 4CL3 (AT1G65060, GeneID: 842814, SEQ ID NO: 3), 4CL5 ( AT3G21230, GeneID: 821677, SEQ ID NO: 4); 4CL derived from Eucalyptus camaldulensis (Genbank Accession No. ACY66928.1, SEQ ID NO: 5) and 4CL (Genbank Accession No. AAZ79469.1, SEQ ID NO: 6); 4CL derived from Galega orientalis (Genbank Accession No. ACZ64784.1, SEQ ID NO: 7); 4CL derived from Pinus massoniana (Genbank Accession No. ACO40513.1, SEQ ID NO: 8) and the like are preferable. These wild-type 4CLs are highly homologous between the amino acid sequences of the carboxylic acid binding pocket, and it is expected that the enzyme activity is greatly shifted by substitution of one amino acid found by the present invention.

The mutant 4CL of the present invention can be easily obtained by introducing the 1 amino acid substitution described above into the wild type 4CL. Preferably, it includes an amino acid sequence into which a substitution as shown below is introduced.
(A1) an amino acid sequence in which the tyrosine residue at position 253 is substituted with a phenylalanine residue or the glutamine residue at position 345 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 1;
(A2) an amino acid sequence in which the tyrosine residue at position 260 is substituted with a phenylalanine residue or the glutamine residue at position 352 is replaced with an alanine residue in the amino acid sequence of SEQ ID NO: 2;
(A3) an amino acid sequence in which the tyrosine residue at position 263 is substituted with a phenylalanine residue or the glutamine residue at position 355 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 3;
(A4) an amino acid sequence in which the tyrosine residue at position 268 is substituted with a phenylalanine residue or the glutamine residue at position 360 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 4;
(A5) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 5 is substituted with a phenylalanine residue or the glutamine residue at position 333 is substituted with an alanine residue;
(A6) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 6 is substituted with a phenylalanine residue, or the glutamine residue at position 333 is substituted with an alanine residue;
(A7) an amino acid sequence in which the tyrosine residue at position 246 is substituted with a phenylalanine residue or the glutamine residue at position 338 is replaced with an alanine residue in the amino acid sequence of SEQ ID NO: 7; or (A8) SEQ ID NO: An amino acid sequence in which the tyrosine residue at position 239 is substituted with a phenylalanine residue in the amino acid sequence of 8, or the glutamine residue at position 331 is replaced with an alanine residue.

A preferred mutant 4CL of the present invention comprises an amino acid sequence into which one amino acid substitution described in the above (A1) is introduced, specifically (A9) the amino acid sequence of SEQ ID NO: 9 or 10 (preferably the amino acid sequence) Consist of). Mutant 4CL consisting of the amino acid sequence of SEQ ID NO: 9 or 10 is referred to as Y253F and Q345A, respectively.

The mutant 4CL of the present invention is
(A10) An amino acid sequence in which substitution mutations, deletions or addition mutations are further introduced in one to several amino acids at positions other than the substitution site in the amino acid sequence of any of (A1) to (A9) above And the protein which consists of the amino acid sequence after the said mutation introduction may contain the amino acid sequence which has dipeptide synthesis ability. Here, the number of amino acids to be substituted, deleted or added is not limited as long as the dipeptide synthesis ability of mutant 4CL is maintained. For example, about 1 to 30, preferably about 1 to 20, more preferably About 1 to 10, even more preferably about 1 to 5, most preferably 1 or 2. The position where amino acid substitution, deletion or addition is performed is not particularly limited as long as the ability to synthesize a dipeptide is maintained. As a preferred specific example, in the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which a tyrosine residue at position 253 is substituted with a phenylalanine residue, and a substitution of an alanine residue is introduced into a glutamine residue at position 345, conversely, Examples of the mutant 4CL include an amino acid sequence in which a glutamine residue at position 345 in the amino acid sequence of SEQ ID NO: 1 is substituted with an alanine residue, and a phenylalanine residue is further substituted into a tyrosine residue at position 253. Similarly, in the amino acid sequences of SEQ ID NOs: 2 to 8, mutant 4CL including an amino acid sequence into which a substitution of 2 amino acids similar to SEQ ID NO: 1 is introduced can be mentioned.

2. Mutant 4CL gene The present invention provides a gene encoding the mutant 4CL (sometimes simply referred to as mutant 4CL gene or mutant 4CL DNA).
In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing genomic DNA and a single-stranded DNA containing cDNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The “gene” or “DNA” includes not only the “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 11 to 20), but also the protein encoded by them and the biological function. Included are “genes” or “DNA” that encode proteins that are equivalent (eg, homologs (such as homologs and splice variants) and derivatives). The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
In the present specification, “polynucleotide” indicates a concept when the above “gene” is viewed from the viewpoint of chemical structure, and “poly” is 2 or more (preferably about 10 to 10,000, more preferably 20 to The number of nucleotides is about 5000), and “nucleotide” includes not only DNA but also RNA.

  The gene encoding wild type 4CL is 4CL2 derived from Arabidopsis thaliana (AT3G21240, GeneID: 821678, SEQ ID NO: 11), 4CL1 (AT1G51680, GeneID: 841593, SEQ ID NO: 12), 4CL3 (AT1G65060, GeneID: 842814, SEQ ID NO: 13). ) 4CL5 (AT3G21230, GeneID: 821677, SEQ ID NO: 14); 4CL derived from Eucalyptus camaldulensis (Genbank Accession No. ACY66928.1, SEQ ID NO: 15) and 4CL (Genbank Accession No. AAZ79469.1, SEQ ID NO: 16); Galega 4CL derived from orientalis (Genbank Accession No. ACZ64784.1, SEQ ID NO: 17); 4CL derived from Pinus massoniana (Genbank Accession No. ACO40513.1, SEQ ID NO: 18) and the like are preferable. These wild-type 4CL-encoding genes have high homology between the nucleotide sequences corresponding to the carboxylic acid binding pockets, and by introducing mutations that enable substitution of one amino acid found by the present invention, The activity is expected to shift greatly.

  The mutant 4CL gene of the present invention can be easily obtained by introducing the mutation that enables substitution of one amino acid as described above into the wild-type 4CL gene. The substitution from tyrosine to phenylalanine includes mutagenesis of a codon (TTT or TTC) encoding phenylalanine to a codon encoding tyrosine (TAT or TAC), preferably point mutagenesis. For the replacement of glutamine with alanine, mutagenesis of a codon encoding alanine (GCT, GCC, GTA or GCG) to a codon encoding glutamine (CAA or CAG) can be mentioned.

Mutagenesis is performed by a known method such as the Kunkel method or the Gapped duplex method or a method similar thereto, for example, using a mutation introduction kit using site-directed mutagenesis, or by using the QuickChange ^™ XL Kit (Stratagene ) And the like.

Preferably, the mutant 4CL gene includes a polynucleotide having a base sequence as shown below.
(B1) In the base sequence of SEQ ID NO: 11, the base sequence encoding tyrosine at positions 757 to 759 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1033 to 1035 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B2) In the base sequence of SEQ ID NO: 12, the base sequence encoding tyrosine at positions 778 to 780 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1054 to 1056 is a base encoding alanine A polynucleotide comprising a base sequence substituted with a sequence;
(B3) In the base sequence of SEQ ID NO: 13, the base sequence encoding tyrosine at positions 787 to 789 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1063 to 1065 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B4) In the base sequence of SEQ ID NO: 14, the base sequence encoding tyrosine at positions 802 to 804 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1078 to 1080 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B5) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 15 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B6) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 16 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B7) In the base sequence of SEQ ID NO: 17, the base sequence encoding tyrosine at positions 736 to 738 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1012 to 1014 encodes alanine A polynucleotide comprising a nucleotide sequence substituted with a nucleotide sequence; or (B8) the nucleotide sequence encoding tyrosine at positions 715 to 717 in the nucleotide sequence of SEQ ID NO: 18 is substituted with a nucleotide sequence encoding phenylalanine, or 991 A polynucleotide comprising a nucleotide sequence obtained by substituting the nucleotide sequence encoding glutamine at position 993 with a nucleotide sequence encoding alanine.

A preferred mutant 4CL gene of the present invention is a polynucleotide comprising a nucleotide sequence introduced with a mutation that enables one amino acid substitution described in (B1) above, specifically (B9) SEQ ID NO: 19 or 20 A polynucleotide comprising the nucleotide sequence of Mutant 4CL genes containing a polynucleotide having the nucleotide sequence of SEQ ID NO: 19 or 20 are referred to as Y253F gene (DNA) and Q345A gene (DNA), respectively.

The mutant 4CL gene of the present invention is
(B10) comprising a polynucleotide comprising a polynucleotide comprising a nucleotide sequence having 70% or more sequence identity to any of the nucleotide sequences of (B1) to (B9), and encoding an enzyme having dipeptide synthesis ability It may be a thing. Such polynucleotides also include degenerate isomers of mutant 4CL genes. Degenerate isomers refer to DNAs that differ only in degenerate codons and can encode the same protein. For example, for a DNA consisting of the nucleotide sequence of SEQ ID NO: 19 or 20, the codon corresponding to any of the amino acids, for example, the codon corresponding to Phe (TTC) has been changed to, for example, TTT which is degenerate. In the present invention, these are called degenerate isomers.

  Here, “70% or more sequence identity” is not limited as long as the ability of the encoded mutant 4CL to synthesize the dipeptide is maintained, but it is about 70% or more, preferably about 80% or more, between base sequences. Preferably, it means about 90% or more, more preferably about 95% or more, and most preferably 98% or more. The identity (%) of the base sequence in the present specification can be determined by using a homology search program (for example, BLAST, FASTA, etc.) commonly used in the art in an initial setting. In the present invention, “identity” of base sequences is calculated by aligning two types of base sequences to be compared and dividing the number of base sequences matched by the alignment by the total number of base sequences as a reference. It is a number showing the percentage in%. Note that the gap generated by the alignment is calculated as a mismatch.

3. Recombinant vector The recombinant vector of the present invention comprises the above-mentioned 2. This gene can be constructed by introducing the gene into an appropriate vector. Here, the vector is preferably selected as appropriate according to the host to be introduced.

  As the plant introduction vector, pBI, pPZP, and pSMA vectors that can introduce a target gene into a plant via Agrobacterium are preferably used. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A binary vector is a shuttle vector that can replicate in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium holding the binary vector, it is a border sequence consisting of LB and RB sequences on the vector. It is possible to incorporate the enclosed DNA into plant nuclear DNA (EMBO Journal, 10 (3), 697-704 (1991)). On the other hand, a pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Also, plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), tobacco mosaic virus (TMV) and the like can be used.

  A baculovirus-derived vector or the like can be used for the insect cell system. In yeast, many vectors containing constitutive promoters such as ADH or LEU2 or inducible promoters such as GAL can be used.

  Examples of vectors for introducing bacteria such as Escherichia coli include the above-described pBI binary vectors and pUC vectors.

  Moreover, those skilled in the art can very easily select appropriate vectors other than these for carrying out the present invention. Depending on the vector used, many suitable transcription and translation elements can be used, such as constitutive or inducible promoters, transcription enhancer elements, transcription terminators, etc., which are known to those skilled in the art. is there.

  The gene of the present invention needs to be incorporated into a vector so that the function of the gene is expressed. Therefore, the vector may contain components such as a promoter, intron, enhancer, translation termination codon, terminator, poly A addition signal, 5′-UTR sequence, selection marker gene, etc. upstream, inside, or downstream of the gene. it can. These can be used in combination with known ones.

  As the promoter, a promoter capable of expressing DNA in a target host may be used. Such promoters are incorporated into various vectors that are commercially available as expression vectors, and can be isolated from the vectors. In addition, components such as introns, enhancers, translation termination codons, terminators, poly A addition signals, 5′-UTR sequences, and selectable marker genes are also incorporated into various vectors that are commercially available as expression vectors. These components can also be isolated from the vector.

  In order to insert a gene into a vector, first, a method in which purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector is employed. .

4). Transformant “Transformation” means that by introducing DNA that is new (foreign) to a cell, the cell undergoes a permanent genetic change. If the cell is a mammalian cell, permanent genetic changes are achieved by introducing DNA into the cell's genome. A “transformant” can be easily obtained by transforming a target host using the recombinant vector.

  When the host is eukaryotic, DNA can be transfected into eukaryotic cells by the calcium phosphate method, microinjection, electroporation, liposome, or viral vector. Also, a eukaryotic cell is transfected with a DNA molecule encoding the protein of the present invention and a second DNA molecule encoding a selectable trait (for example, a herpes simplex virus thymidine kinase gene). Transformants can be selected. Transformed eukaryotic cells can express the protein of the present invention by culturing under a predetermined condition in a medium suitable for the growth of the cells.

  Examples of host cell strains that can be used as mammalian cells include CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, WI38, and the like. Examples of host cell strains that can be used as insect cells include Sf9. Examples of host cell strains that can be used as yeast cells include Saccharomyces cerevisiae.

  When the host is a prokaryotic cell (for example, E. coli), competent cells having the ability to take up DNA can be prepared by treating with the calcium chloride method by procedures well known to those skilled in the art. Transformation can also be performed by alternative methods such as electroporation. A prokaryotic cell together with a DNA molecule encoding the protein of the present invention and a second DNA molecule encoding a selectable trait (for example, a gene resistant to antibiotics such as ampicillin and kanamycin). Transfectants can be selected by transfection. The transformed prokaryotic cell can be cultured in a medium suitable for the growth of the cell (preferably in the presence of an antibiotic) under predetermined conditions to express the protein of the present invention.

  The mutant 4CL of the present invention is preferably obtained by expressing a DNA encoding the mutant 4CL in a prokaryotic organism using the recombinant vector of the present invention. For example, when E. coli is used as a host, mutant 4CL can be expressed by culturing E. coli on a large scale.

  Regarding the isolation or purification technique after expressing the mutant 4CL of the present invention, immunology using conventionally known means such as chromatographic separation, ammonium sulfate precipitation, and antigen or antibody (monoclonal antibody, polyclonal antibody). Can be performed by conventional separation methods.

  For example, purification of the mutant 4CL of the present invention from microorganisms such as Escherichia coli can be simplified by using nickel chelate chromatography and providing a label for purification in one step in the protein sequence. For example, a polyhistidine label composed of a plurality (preferably 5 to 15) histidine residues is incorporated into the amino terminus or carboxyl terminus of the mutant 4CL of the present invention, labeled with polyhistidine, and subjected to nickel chelate chromatography. It is possible to efficiently perform protein isolation in one step. The mutant 4CL of the present invention can be designed to include a suitable enzyme cleavage site for the purpose of improving recovery.

5). Transformed plant body 3. Using the recombinant vector prepared in (1) above, the transformed plant body carrying the mutant 4CL of the present invention can be prepared by transforming and regenerating the cells of the target plant.

  In preparing a transformed plant body, various methods already reported and established can be used as appropriate, and preferable examples thereof include, for example, biological methods such as viruses and Agrobacterium. Examples include methods using Ti plasmids, Ri plasmids and the like as vectors. Physical methods include electroporation, polyethylene glycol, particle gun, microinjection (Plant Genetic Transformation and Gene Expression; a laboratory manual, J. Draper et al. al., Blackwell Scientific Publication (1988)), Silicon Whisker (Euphytica, 85, 75-80 (1995); In Vitro Cell. Dev. Biol., 31, 101-104 (1995); Plant Science, 132, 31 -43 (1998)), liposomes, vacuum infiltration (CR Acad. Sci. Paris, Life Science, 316: 1194 (1993)), etc. Can be mentioned. The introduction method can be appropriately selected and used by those skilled in the art.

  In general, a gene introduced into a plant is integrated into the genome of the host plant, but in this case, a phenomenon called a position effect, in which the expression of the transgene is different due to different positions on the introduced genome, is seen. Confirmation of the transgene is necessary.

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. After performing PCR, the amplified product is subjected to electrophoresis such as agarose gel electrophoresis, stained with ethidium bromide solution, etc., and the amplified product is detected as a single band to confirm that it has been transformed. Can be confirmed.

  Examples of the plant used for transformation in the present invention include cells such as monocotyledonous plants such as rice and taro, dicotyledonous plants such as Arabidopsis and tobacco, and particularly preferred plant types include Arabidopsis and tobacco. It is done.

  In the present invention, the plant material to be transformed includes, for example, growth point, shoot primordia, meristem, leaf piece, stem piece, root piece, tuber piece, petiole piece, protoplast, callus, bud, pollen, pollen Examples include cells such as tubes, flower pattern pieces, flower stem pieces, petals, and sepals.

  In the case of targeting plant cells, a known tissue culture method may be used to regenerate a transformant from the obtained transformed cells. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. For regeneration from plant cells to plant bodies, for example, references such as “Plant Cell Culture Manual” (Yasuyuki Yamada, Kodansha Scientific, 1984) can be referred to.

  The transformed plant of the present invention is not only the “T0 generation”, which is a regenerated generation that has undergone transformation treatment, but also the “T1 generation”, a progeny obtained from seeds of self-propagation or other breeding of the plant, Progeny plants such as the next generation (T2 generation) obtained by self-propagation or other breeding of "T1 generation" plants that have been found to be transgenic by selection or analysis by the Southern method, etc. It also includes generations and individuals that can be obtained by any means of cultivation and breeding based on the T1 generation, such as individuals that have maintained growth, and mutant individuals whose specific traits have changed from the next generation such as the T1 generation.

  The transformed plant body is cultivated under conditions suitable for the plant body and conditions suitable for the production of the amide compound (for example, a substrate containing a carboxylic acid as a substrate and a substrate containing an amine are given at appropriate concentrations). The target amide compound can be obtained in high yield by harvesting and recovering the product.

6). Method for Producing Compound Having Amide Bond Using Mutant 4CL In one embodiment, a compound having an amide bond (hereinafter referred to as amide) is obtained by reacting the isolated mutant 4CL with a substrate in the following reaction step in vitro. May be abbreviated as compound).

(6-1) A step of reacting the mutant 4CL of the present invention in the presence of a carboxylic acid-containing substrate, an amine-containing substrate, and ATP The carboxylic acid-containing substrate and the amine-containing substrate depend on the desired amide compound. Thus, an appropriate combination of substrates can be selected as appropriate. Unlike the wild type 4CL, the mutant type 4CL can generate polyamino acids (that is, dipeptides or polypeptides) having various amide bonds using amino acids as substrates. Therefore, the substrate containing carboxylic acid and the substrate containing amine are preferably amino acids that are substrates containing carboxylic acid and amine per molecule. The amino acid may be either an L-amino acid or a D-amino acid, and 20 kinds of L-amino acids constituting a natural protein, alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), Phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), Examples include arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y). In addition to the 20 amino acids, selenocysteine (U) or pyrrolidine (O ) And the like, as well as their D-amino acids. Other examples include homoserine, β-alanine, sarcosine, ornithine, creatine, γ-aminobutyric acid, and opain.

The reaction conditions of mutant 4CL are not particularly limited as long as an amide bond is formed from the substrate, but divalent ions such as Ca ⁺⁺ , Mg ⁺⁺ , Mn ⁺⁺ (preferably Mg ⁺⁺ ). In a buffer solution having a pH of 6 to 9 containing 0.1 to 10 mM of ATP and a predetermined concentration (for example, 0.1 to 10 mM) of a substrate, 20 to 50 ° C. and 0.1 to 24 hours are exemplified.

  After completion of the reaction, the amide compound produced from the reaction solution is recovered and purified according to the purpose. Recovery and purification of the amide compound can be performed using means well known in the art.

  In another embodiment, a compound having an amide bond can be produced by culturing the transformant of the present invention in the presence of a substrate.

(6-2) Step of culturing the transformant in the presence of a carboxylic acid-containing substrate, an amine-containing substrate, and ATP. The transformant used is the growth rate of the transformant, the expression of mutant 4CL, and the enzyme reaction. From the viewpoint of ease of setting the conditions, a transformant can be selected according to the purpose. A suitable example is an E. coli transformant. Hereinafter, E. coli will be described as an example.

  The substrate containing carboxylic acid and the substrate containing amine are as described in (6-1) above.

Mutant 4CL expresses transformed E. coli as a foreign gene product in culture as an exogenous gene product in the E. coli, and by continuing the culture, the enzyme reaction proceeds using the substrate in the medium, and the reaction product is It is expected to accumulate in E. coli or be released outside the cell. The reaction conditions are not particularly limited, but a culture solution having a pH of 6 to 9 containing divalent ions (preferably Mg ⁺⁺ ) such as Ca ⁺⁺ , Mg ⁺⁺ , Mn ⁺⁺ (for example, LB (Luria -Bertani) medium) is exemplified in the presence of a substrate at a predetermined concentration (for example, 1 to 1000 mM) at 20 to 40 ° C. for 24 to 90 hours. The culture can be scaled up using a bioreactor or the like and can be continued continuously.

  After completion of the reaction, the produced amide compound is recovered from the culture or culture medium and purified according to the purpose. Recovery and purification of the amide compound can be performed using means well known in the art.

7.4 Method for producing compound having amide bond using 4CL In the present invention, 4CL can catalyze the synthesis of coumaric acid amide and amide of specific carboxylic acid other than coumaric acid in addition to the synthesis of thioester of coumaric acid. It was found. The present invention also provides a method for producing an amide compound using isolated 4CL.

4. A step of reacting coumaroyl CoA synthase in the presence of coumaric acid or other carboxylic acid, amine-containing substrate and ATP in the absence of CoASH, and carboxylic acids other than coumaric acid include cinnamic acid, sinapinic acid, ferulic acid, etc. Acids similar to coumaric acid. The aforementioned amino acids (eg, glutamic acid, aspartic acid) can also be used.

  The substrate containing an amine is an amine containing an amino acid, preferably a primary amine. For example, the various amino acids described above may be used. When an amino acid is used as a substrate containing an amine in this step, an amide bond is formed between a carboxyl group contained in coumaric acid or other carboxylic acid and an amino group contained in the amino acid.

  The term “in the absence of CoASH” is not particularly limited as long as the formation of a thioester with a carboxylic acid by CoASH does not proceed, but it is highly possible that CoASH is present in vivo, so in vitro (in vitro ) In the absence of CoASH.

The reaction condition of 4CL is not particularly limited as long as an amide bond is formed from the substrate, but includes divalent ions (preferably Mg ⁺⁺ ) such as Ca ⁺⁺ , Mg ⁺⁺ , and Mn ^++. Examples are 0.1 to 24 hours at 20 to 45 ° C. in the presence of 0.1 to 2 mM ATP and a substrate having a predetermined concentration (for example, 0.1 to 5 mM) in a pH 6 to 9 buffer.

  After completion of the reaction, the amide compound produced from the reaction solution is recovered based on the characteristics of the compound and purified. Recovery and purification of the amide compound can be performed using means well known in the art.

  Examples of the amide compound synthesized by this production method include amide of coumaric acid and amino acid; theanine synthesized from L-glutamic acid and ethylamine; capsaicin; homoserine lactones, etc., but are limited to these compounds. It is not a thing.

  The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Example 1 RNA Extraction and cDNA Preparation Total RNA was extracted from 5 g of Arabidopsis thaliana leaves using RNeasy Plant Mini Kit (QIAGEN). A reverse transcription reaction was performed using Super dR primer with SuperScript ^R II Reverse Transcriptase (Invitrogen) to prepare cDNA.

Example 2 Cloning of 4-Coumaroyl-CoA ligase 2 (At3g21240) from Arabidopsis thaliana
It was amplified target sequence by PrimeSTAR ^R HS DNA Polymerase PCR method using and the following primers. The amplification product was electrophoresed in 1.2% agarose gel, cut out 1.6 kbp band is the size of the object, and purified using ^{QIAquick R Gel Extraction Kit (QIAGEN)} , perform ethanol precipitation, and the insert DNA.
A primer manufactured by Invitrogen was used.
5'-ACG TAC GCA TGC ATG ACG ACA CAA GAT GTG ATA G-3 '(SphI restriction site is underlined) (SEQ ID NO: 21)
5'-GGT CAG CTG CAG CTA GTT CAT TAA TCC ATT TGC TAG-3 '(PstI restriction site is underlined) (SEQ ID NO: 22)
The purified DNA and pQE80L (QIAGEN) were treated with restriction enzymes from SphI and PstI, and purified by the above DNA purification method. Using DNA Ligation Kit Ver.2.1 (TAKARA), a plasmid DNA for enzyme expression having a 6x histidine tag added to the N-terminus was prepared and transformed into E. coli M15 [pREP4] (QIAGEN).

Example 3 Protein Expression by E. coli The E. coli transformed in Example 2 was cultured with shaking in 1000 mL of Luria-Bertani medium containing 100 mg of Ampicillin until OD600 = 0.6. Isopropylthio-β-D-galactoside (IPTG) was added to the medium to a final concentration of 1 mM, and further cultured with shaking at 23 ° C. for 12 hours.

Example 4 Protein Extraction and Purification After collecting the enzyme-induced Escherichia coli cultured in Example 3, it was suspended in 40 mM potassium phosphate buffer (KPB) containing 0.1 M NaCl and 5 mM imidazole, pH 7.9, using a sonicator. E. coli cells were disrupted. The crushed material was centrifuged at 10,000 rpm, 4 ° C., 30 min, and the supernatant was used as a crude protein extract.
The obtained crude extract was subjected to Ni Sepharose ^™ 6 Fast Flow (GE Healthcare), washed with 40 mM KPB containing 0.5 M NaCl and 40 mM imidazole, and then 15 mM KPB containing 10% (w / v) glycerol and 500 mM imidazole ( Elution was performed at pH 7.5) to obtain a purified enzyme. The purified enzyme solution was desalted and concentrated using 0.1 mM HEPES (pH 7.0) containing 5% (w / v) glycerol and 0.2M NaCl and Amicon Ultra-4 (Ultracel-10k).

Example 5 AMP Assay 37 μm in 100 μl of 0.1 mM Tris-HCl (pH 7.5) containing 2 mM MgCl ₂ with 0.2 mM ATP using 0.8 μm enzyme, 0.8 mM coumaric acid or other carboxylic acid and 0.8 mM CoASH or amino acid as substrate The reaction was carried out at 5 ° C. for 5 minutes. After adding 16 mM trichloroacetic acid (TCA) to the enzyme reaction solution and stirring, the mixture was centrifuged at 15,000 rpm for 1 min, and the supernatant was measured with an ACQUITY ^™ UPLC system (Waters). The measurement conditions are as follows.
Column: ACQUITY UPLC HSS T3 1.8 μM (2.1mmI.D. x 50mm)
Flow rate: 0.19 mL / min
Eluent: 0.1% phosphate buffer and acetonitrile (AcCN)
0-3min, 0% AcCN; 3-7min, 0-100% AcCN; 7-12min, 100% AcCN
Detection: UV = 260 nm.

Example 6 Identification of Enzyme Reaction Product 100 μg enzyme, 0.1 mM coumaric acid or other carboxylic acid (8-methyl-6-nonenoic acid) and 0.5 mM CoASH or amino acid or amine (4-hydroxy-3-methoxybenzyl) as substrate Amine) and 0.1 mM Tris-HCl (pH 7.5) containing 2 mM MgCl ₂ together with 0.2 mM ATP was reacted at 30 ° C. for 8 hours. After adding 32 mM TCA to the enzyme reaction solution and stirring, the mixture was centrifuged at 15,000 rpm for 1 min, and the supernatant was measured by LC-ESI MS using Agilent Technologies HPLC 1100 series and Bruker Daltonics esquire4000. The measurement conditions are as follows.
Column: CAPCELL PK C8 DD (4.6mmI.D. x 150mm)
Flow rate: 0.2 mL / min
Eluent: 1% acetic acid in water and methanol (MeOH)
0-15min, 20-60% MeOH; 15-25min, 60-70% MeOH; 25-32min, 70-80% MeOH; 32-40min, 80-100% MeOH; 40-55min, 100% MeOH
Detection: UV = 290 nm.

Example 7 Production of Mutant Enzyme
Mutation was introduced by PCR using QuickChange ^™ XL Kit (Stratagene) with the following primers. A primer manufactured by Invitrogen was used.
Q345A Fw: 5'-GCTAAGTTTCCTAACGCCAAGCTTGGTGCGGGCTATGGGATG-3 '(SEQ ID NO: 23)
Q345A Rv: 5'-CCTGCTTCTGTCATCCCATAGCCCGCACCAAGCTTGGCG-3 '(SEQ ID NO: 24)
Y253F Fw: 5'-TTGCCTATGTTCCATATATTCGCTCTCAACTCCATCAT-3 '(SEQ ID NO: 25)
Y253F Rv: 5'-AGAGCATGAGGAGTTGAGAGCGAATATATGGAACATA-3 '(SEQ ID NO: 26)
According to Examples 2 to 4, transformation and enzyme purification were performed in the same manner as in the wild type and used for the enzyme reaction.

Example 8 Dipeptide Synthesis Reaction Using 500 μg of enzyme and 0.4 mM L-phenylalanine as a substrate, the reaction was carried out at 30 ° C. for 24 hours in 100 μl of 0.1 mM Tris-HCl (pH 7.5) containing 2 mM MgCl ₂ together with 0.2 mM ATP. The enzyme reaction solution was adjusted to about pH 9 with sodium hydrogen carbonate, 1 mM Dansyl chloride was added, reacted at 60 ° C. for 1 hour, and adjusted to about pH 1 with HCl. After the adjustment, the reaction solution was extracted with ethyl acetate, dried, and measured by LC-ESI MS using Agilent Technologies HPLC 1100 series and Bruker Daltonics esquire4000. The measurement conditions are as follows.
Column: CAPCELL PK C8 DD (4.6mmI.D. x 150mm)
Flow rate: 0.2 mL / min
Eluent: Water containing 1% acetic acid and acetonitrile (AcCN)
0-20min, 40-70% AcCN; 20-40min, 70% AcCN; 40-60min, 70-100% AcCN; 60-100min, 100% AcCN
Detection: UV = 360 nm.

(1) Examination of substrate specificity and expansion of catalytic function from CoA thioester to amide ligase Arabidopsis thaliana-derived 4CL (At4CL2) is a soluble protein consisting of a monomer with a molecular weight of 60 kDa that can be expressed heterologously in E. coli. (Stuible, H.-P., Kombrink, E. (2001) Identification of the substrate specificity-conferring amino acid residues of 4-coumarate: coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties, J. Biol. Chem. 276, 26893-26897; Schneider, K., Hovel, K., Witzel K., Hamberger, B., Schomburg, D., Kombrink, E., Stuible H.-P. (2003) The substrate specificity-determining amino acid code of 4-coumarate: CoA ligase, Proc. Natl. Acad. Sci. USA 100, 8601-8606). Based on a homology model based on the crystal structure of the adenylation domain of gramicidin S synthase (NRPS) as a template, the specificity for the initiation substrate is changed by introducing site-specific mutations into the initiation substrate carboxylic acid binding pocket. However, although it has been reported that cinnamic acid and sinapinic acid are activated instead of coumaric acid (Schneider, K., et al., Proc. Natl. Acad. Sci. USA 100, 8601-8606 ( 2003)), there is no report on the specificity of the thiol side substrate. Therefore, first, using a recombinant enzyme heterologously expressed and purified in Escherichia coli, 20 kinds of L-amino acids together with coumaric acid and ATP were allowed to act as substrates instead of CoASH, and the enzyme activity was examined. As a result, it was surprisingly found that all amino acids function as substrates as in the case of the original CoASH, and generate amides with coumaric acid (FIG. 2). As a result of measuring enzyme reaction kinetics, it was shown that the amide synthase reaction proceeds with higher efficiency than the original production of CoA thioester. On the other hand, more surprisingly, as a result of detailed analysis of the enzymatic reaction product using L-phenylalanine, another molecule of phenylalanine was condensed in addition to coumaroyl-L-Phe in which phenylalanine was amide-bonded to coumaric acid. Production of coumaroyl-L-Phe-L-Phe was also observed (FIG. 3). In addition, D-form amino acid showed almost the same activity and produced an amide with coumaric acid.

  Next, as a result of using coumaric acid or hexanoic acid as the starting substrate and homoserine as the amino substrate, amide and homoserine lactone responsible for quorum sensing of microorganisms were successfully produced. In addition, capsaicin, a pungent component of chili pepper, could be produced in the same manner (FIG. 4). Thus, the tolerant substrate specificity and potential catalytic ability, which can be said to be an exceptional example of 4CL, deserve special mention. In at least in vitro reactions, as long as the adenylation of the carboxyl group proceeds, in addition to the thioester CS bond with the original CoA, a wide range of amines as substrates (particularly low substrate specificity for amines) It was found to be a multifunctional enzyme that also catalyzes formation. In the model plant Arabidopsis thaliana, in addition to 3 isozymes of 4CL, there are 25 or more 4CL homologous enzymes exhibiting at least 25% sequence homology, and about 200 adenylating enzyme genes having an AMP binding site. At present, most of the functions are unidentified, and the role in the biosynthesis of plant secondary metabolites is very interesting.

(2) Introduction of point mutations into the starting substrate binding pocket and expansion of functions to peptide synthase As described above, what is important in the enzymatic reaction of 4CL is the adenylation of the carboxyl group of the starting substrate, and this substrate specificity If this can be controlled, the enzyme catalytic function can be further expanded. Therefore, the present inventors newly constructed a homology model using luciferase as a template (FIG. 5), and introduced site-specific mutations for Y253 and Q345 among the amino acid residues constituting the 4CL carboxylic acid binding pocket. The effects on enzyme activity were investigated. As a result, significant enhancement of CoA synthesis activity was observed with substitutions such as Y253F, Y253A, and Q345A (FIG. 6). In addition, when using an artificial substrate such as indole acrylic acid in which the aromatic ring of coumaric acid was substituted with indole, the activity was increased by these mutant enzymes, and a new non-natural CoA thioester was produced.

  On the other hand, the 4CL wild type enzyme cannot accept an amino acid as an initiation substrate, and formation of a thioester or amide always proceeds using coumaric acid as an initiation substrate. Surprisingly, however, in the Y253F and Q345A mutant enzymes described above, in the absence of coumaric acid, the enzymatic reaction proceeds only with amino acid and ATP as a substrate, and after formation of aminoacyl AMP, another molecule of It was found that a dipeptide was produced in a high yield by condensation with an amino acid (FIG. 6). Dipeptide synthesis activity is remarkable with L-phenylalanine and L-leucine among 20 kinds of amino acids, and the reaction efficiency exceeds the original coumaroyl CoA synthesis activity by 50% or more (FIGS. 6 and 7). . That is, by replacing a single amino acid residue in the starting substrate binding pocket of wild type 4CL, a catalytic function as a supernatural dipeptide synthase was newly acquired in addition to the original CoA synthesis activity.

(3) Development of enzyme inhibitor and elucidation of enzyme three-dimensional structure by X-ray crystal structure analysis No report has been made on the X-ray crystal structure analysis of 4CL. As described above, enzymes belonging to the ANL superfamily have a characteristic loop structure at the C-terminus, and it is known that the C-terminal domain undergoes a large dynamic structural change as the enzymatic reaction proceeds. (FIG. 5) (Gulick, AM (2009) Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase, ACS Chem. Biol. 4, 811-827). Such a structural change has great significance for the formation of the above-described thioester, amide bond, and dipeptide. Therefore, in order to establish the structural basis of this enzyme reaction mechanism, X-ray crystal structure analysis of 4CL wild-type and mutant enzymes and development of enzyme inhibitors used for co-crystals are underway.
First, as an inhibitor, a transition state intermediate analog of coumaric acid adenylate (FIG. 8) was chemically synthesized, and it was shown that this compound inhibits the enzyme reaction at the nM level as expected. On the other hand, we succeeded in crystallizing recombinant enzymes heterologously expressed in Escherichia coli and are currently working on refinement.

  The method for producing an amide compound using the mutant 4 coumaroyl CoA synthetase of the present invention can efficiently produce dipeptides, bioactive amides and the like under clean and mild conditions as compared with the organic synthesis method. The mutant 4 coumaroyl CoA synthetase and the production method using the same of the present invention can be used as various raw materials for pharmaceuticals, cosmetics, and foods (functional foods) by selecting appropriate carboxylic acids and amines as substrates. Amide compounds can be provided.

Claims (14)

  A mutant type 4 coumaroyl CoA synthase in which at least one amino acid in an amino acid sequence constituting a carboxylic acid binding pocket of 4 coumaroyl CoA synthase is substituted, and at least dipeptide synthesis ability is obtained by the substitution.   The mutant 4 coumaroyl CoA synthase according to claim 1, wherein the substitution is a substitution from a tyrosine residue to a phenylalanine residue or a substitution from a glutamine residue to an alanine residue. The mutant 4 coumaroyl CoA synthetase according to claim 1 or 2, comprising the following amino acid sequences (A1) to (A10):
(A1) an amino acid sequence in which the tyrosine residue at position 253 is substituted with a phenylalanine residue or the glutamine residue at position 345 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 1;
(A2) an amino acid sequence in which the tyrosine residue at position 260 is substituted with a phenylalanine residue or the glutamine residue at position 352 is replaced with an alanine residue in the amino acid sequence of SEQ ID NO: 2;
(A3) an amino acid sequence in which the tyrosine residue at position 263 is substituted with a phenylalanine residue or the glutamine residue at position 355 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 3;
(A4) an amino acid sequence in which the tyrosine residue at position 268 is substituted with a phenylalanine residue or the glutamine residue at position 360 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 4;
(A5) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 5 is substituted with a phenylalanine residue or the glutamine residue at position 333 is substituted with an alanine residue;
(A6) an amino acid sequence in which the tyrosine residue at position 241 in the amino acid sequence of SEQ ID NO: 6 is substituted with a phenylalanine residue, or the glutamine residue at position 333 is substituted with an alanine residue;
(A7) an amino acid sequence in which the tyrosine residue at position 246 is substituted with a phenylalanine residue in the amino acid sequence of SEQ ID NO: 7 or the glutamine residue at position 338 is substituted with an alanine residue;
(A8) an amino acid sequence in which the tyrosine residue at position 239 is substituted with a phenylalanine residue or the glutamine residue at position 331 is substituted with an alanine residue in the amino acid sequence of SEQ ID NO: 8;
(A9) the amino acid sequence of SEQ ID NO: 9 or 10; or (A10) at a position other than the substitution site of any one of the amino acid sequences of (A1) to (A9), further one to several amino acids are substituted or missing. An amino acid sequence into which a loss or addition mutation has been introduced, wherein the protein comprising the amino acid sequence after the mutation is capable of synthesizing a dipeptide.   A gene encoding the mutant type 4 coumaroyl CoA synthase according to any one of claims 1 to 3. The gene according to claim 4, comprising the following polynucleotides (B1) to (B10):
(B1) In the base sequence of SEQ ID NO: 11, the base sequence encoding tyrosine at positions 757 to 759 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1033 to 1035 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B2) In the base sequence of SEQ ID NO: 12, the base sequence encoding tyrosine at positions 778 to 780 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1054 to 1056 is a base encoding alanine A polynucleotide comprising a base sequence substituted with a sequence;
(B3) In the base sequence of SEQ ID NO: 13, the base sequence encoding tyrosine at positions 787 to 789 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1063 to 1065 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B4) In the base sequence of SEQ ID NO: 14, the base sequence encoding tyrosine at positions 802 to 804 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1078 to 1080 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B5) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 15 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B6) The base sequence encoding tyrosine at positions 721 to 723 in the base sequence of SEQ ID NO: 16 is substituted with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 997 to 999 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B7) In the base sequence of SEQ ID NO: 17, the base sequence encoding tyrosine at positions 736 to 738 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 1012 to 1014 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B8) In the base sequence of SEQ ID NO: 18, the base sequence encoding tyrosine at positions 715 to 717 is replaced with the base sequence encoding phenylalanine, or the base sequence encoding glutamine at positions 991 to 993 encodes alanine A polynucleotide comprising a base sequence substituted with the base sequence;
(B9) a polynucleotide comprising the base sequence of SEQ ID NO: 19 or 20, or (B10) a polynucleotide comprising a base sequence having 70% or more sequence identity to any of the base sequences (B1) to (B9) above A polynucleotide encoding an enzyme having the ability to synthesize dipeptides.   A recombinant vector containing the gene according to claim 4 or 5.   A transformant into which the recombinant vector according to claim 6 has been introduced.   A method for producing a compound having an amide bond, comprising the step of reacting the mutant type 4 coumaroyl CoA synthetase according to any one of claims 1 to 3 in the presence of a substrate containing a carboxylic acid, a substrate containing an amine, and ATP. .   A method for producing a compound having an amide bond, comprising culturing the transformant according to claim 7 in the presence of a substrate containing a carboxylic acid, a substrate containing an amine, and ATP.   The production method according to claim 8 or 9, wherein the substrate containing carboxylic acid and the substrate containing amine are amino acids, and the compound having an amide bond is a peptide.   4. A method for producing a compound having an amide bond, comprising a step of reacting coumaroyl CoA synthase with coumaric acid or other carboxylic acid, a substrate containing an amine, and ATP in the absence of CoASH.   The production method according to claim 11, wherein the compound having an amide bond is coumaric acid amide.   The production method according to claim 11, wherein the compound having an amide bond is capsaicin.   The production method according to any one of claims 11 to 13, wherein the 4 coumaroyl CoA synthase is a wild-type 4 coumaroyl CoA synthase.