Method for producing n-acyl-amino group-containing compound

Abstract

To provide a method of efficiently producing an N-acyl-amino group-containing compound by an enzymatic method.SOLUTION: The present invention provides a method of producing an N-acyl-amino group-containing compound, comprising producing the N-acyl-amino group-containing compound by reacting an amino group-containing compound with a carboxyl group-containing compound, in the presence of an enzyme having the ability to bond a carboxyl group and an amino group in an ATP dependent manner to form an amide bond.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an N-acyl-amino group-containing compound. [Background technology]

[0002] N-acyl-amino group-containing compounds (e.g., Nα-acylamino acids) are used as cosmetic ingredients (e.g., surfactants) and the like. Chemical synthesis of N-acyl-amino group-containing compounds (e.g., Schotten-Baumann reaction) has the problem of environmental burden due to by-products of the synthesis reaction. Therefore, enzymatic synthesis of N-acyl-amino group-containing compounds is required. Several prior arts regarding the enzymatic synthesis of N-acyl-amino group-containing compounds have been reported.

[0003] Patent Document 1 reports Nα-acylamino acid fermentation from sugar using the surfactin biosynthesis enzyme of Bacillus subtilis. However, since the amount of Nα-acylglutamic acid produced is only 116.8 mg / L, this fermentation is not suitable for industrial-scale production.

[0004] Patent Document 2 reports a method for synthesizing Nα-acylglycine from amino acids and fatty acids using human amino acid N-acyltransferase and E. coli acyl-CoA synthetase. However, this method cannot directly bind amino acids to fatty acids and requires a two-step enzyme reaction, which makes the control more complicated than reactions using a single enzyme.

[0005] Non-Patent Document 1 reports a method for synthesizing Nα-acylamino acids from amino acids and fatty acids in a solution containing glycerol using acylase derived from pig kidney. This method utilizes the fact that the hydrolysis reaction of Nα-acylamino acids by acylase does not proceed easily in a solution containing glycerol. However, in view of the requirement for the use of a large amount of glycerol and the fact that the synthesis of Nα-acylamino acids in an aqueous solvent not containing glycerol results in a low yield, this method is inefficient for industrial production.

[0006] Non-Patent Document 2 reports a method for synthesizing Nα-acylamino acids from amino acids and fatty acids in a solution containing glycerol using acylase derived from Streptomyces mobaraensis. However, since there has been no report on the synthesis of Nα-acylamino acids in an aqueous solvent not containing glycerol, the efficiency of this method for industrial production has not been demonstrated. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2008 / 131002 [Patent Document 2] International Publication No. 2015 / 028423 [Non-patent literature]

[0008] [Non-Patent Document 1] Wada et al., Journal of the American Oil Chemists' Society, 2002, 79(1), pp 41-46 [Non-Patent Document 2] Koreishi et al., Journal of Agricultural and Food Chemistry, 2006, 54(1), pp 72-78 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an efficient method for producing an N-acyl-amino group-containing compound by an enzymatic method. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have found that an enzyme having the ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner can efficiently produce N-acyl-amino group-containing compounds from carboxyl group-containing compounds and amino group-containing compounds, including fatty acids, and have thus completed the present invention.

[0011] That is, the present invention is as follows. [1] A method for producing an N-acyl-amino group-containing compound, comprising reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of an enzyme capable of combining a carboxyl group and an amino group in an ATP-dependent manner to form an amide bond, to produce an N-acyl-amino group-containing compound. [2] The method according to [1], wherein the enzyme is an enzyme derived from a plant or a microorganism. [3] The method according to [1] or [2], wherein the enzyme is a GH3 protein. [4] The method according to any one of [1] to [3], wherein the enzyme is a GH3 protein belonging to any one of group I, group II, or group III. [5] The GH3 protein is selected from the group consisting of the following: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, which contains one or several amino acid substitutions, deletions, insertions, or additions, and which has N-acylase activity; or (C) a protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 and having N-acylase activity; Any of the methods according to [1] to [4], selected from the group consisting of: [6] The method according to [1] or [2], wherein the enzyme is a PaaK protein. [7] The PaaK protein is selected from the group consisting of the following: (A') a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11; (B') a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 10 or 11 and having N-acylase activity; or (C') a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 or 11 and having N-acylase activity; The method according to [6], wherein the compound is selected from the group consisting of [8] The method according to any one of [1] to [7], wherein the amino group-containing compound is an amino group-containing compound having an anionic group. [9] The method according to any one of [1] to [8], wherein the amino group-containing compound is an amino acid or a peptide.

[10] The method according to [9], wherein the amino group-containing compound is an α-amino acid, β-amino acid, or γ-amino acid, or a dipeptide thereof.

[11] The method according to [9] or

[10] , wherein the amino acid is an L-amino acid or a D-amino acid.

[12] The amino group-containing compound is selected from the group consisting of the following: (1) (a) an α-amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine; (b) β-alanine; (c) gamma-aminobutyric acid; and (d) sarcosine; an amino acid selected from the group consisting of: (2) taurine; and (3) a dipeptide selected from the group consisting of aspartylphenylalanine, glycylglycine, and alanylhistidine; Any of the methods according to [1] to

[11] , which is selected from the group consisting of:

[13] The method according to any one of [1] to

[12] , wherein the carboxyl group-containing compound is a fatty acid.

[14] The method according to

[13] , wherein the fatty acid is a fatty acid having 6 to 18 carbon atoms.

[15] The method according to

[14] , wherein the fatty acid is a fatty acid having 6 to 12 carbon atoms.

[16] The method according to

[14] or

[15] , wherein the fatty acid is a saturated fatty acid.

[17] Any of the methods according to [1] to

[16] , wherein the enzyme is a purified enzyme.

[18] The method according to any one of [1] to

[17] , wherein the reaction in the presence of the enzyme is carried out using a transformed microorganism that produces the enzyme, or a processed product thereof.

[19] The method according to

[18] , wherein the transformed microorganism is any one of the following microorganisms (i) to (iii): (i) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto; (ii) a microorganism comprising, in a non-native genomic region or in a non-genomic region, an expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto; or (iii) A microorganism comprising an expression unit containing a polynucleotide encoding the enzyme in multiple copies.

[20] The method according to

[18] or

[19] , wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae.

[21] The method according to

[20] , wherein the bacterium is Escherichia coli. Effect of the Invention

[0012] According to the method of the present invention, the reaction for producing an N-acyl-amino group-containing compound by amide bonding between an amino group-containing compound and a carboxyl group-containing compound can be carried out efficiently. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides a method for producing an N-acyl-amino group-containing compound. The method of the present invention comprises reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of an enzyme to produce the N-acyl-amino group-containing compound.

[0014] The enzymes used in the methods of the invention have the ability to combine carboxyl and amino groups in an ATP-dependent manner to form an amide bond. It is believed that the enzymes used in the methods of the invention activate a carboxyl-containing compound by adenylation, and form the amide bond by a mechanism in which the amino-containing compound nucleophilically attacks the adenylated intermediate.

[0015] The enzyme used in the method of the present invention may be derived from a plant or a microorganism. Examples of plants from which the enzyme used in the method of the present invention is derived include plants belonging to Gymnosperms, Angiosperms, Pteridophytes, Lycopods, Anthoceros, Marginale, Charophyceae, Zygophyceae, Chlorophyta, Glaucophyta, and Rhodophyta. More specifically, Arabidopsis (e.g., Arabidopsis thaliana), Oryza (e.g., Oryza sativa), Capsicum (e.g., Capsicum chinense), Glycine (e.g., Glycine max), Solanum or Lycopersicon (e.g., Solanum lycopersicum or Lycopersicon esculentum), Nicotiana (e.g., Nicotiana tabacum), Physcomitrella (e.g., Physcomitrella patens), Citrus (e.g., Citrus madurensis), Pinus (e.g., Pinus pinaster), Brassica (e.g., Brassica napus), Gossypium sp., Vitis (e.g., Vitis vinifera), Medicago (e.g., Medicago truncatula), Populus, Triticum (e.g., Triticum aestivium), Zea (e.g., Zea mays), Hordeum (e.g., Hordeum vulgare), and Sorghum (e.g., Sorghum bicolor).Microorganisms from which the enzymes used in the method of the present invention are derived include those of the genus Cystobacter (e.g., Cystobacter fuscus), Synechococcus (e.g., Synechococcus sp.), Pantoea (e.g., Pantoea agglomerans), and Pseudomonas (e.g., Pseudomonas savastanoi).

[0016] The enzyme used in the method of the present invention may be a GH3 protein. The term "GH3 protein" refers to a group of enzymes that function in the amidation of carboxyl-containing plant hormones, such as jasmonic acid, auxins (indole-3-acetic acid), salicylic acid, and substituted benzoates, and their homologs. As a structural feature, the term "GH3 protein" refers to a protein that contains a GH3 superfamily domain. The GH3 superfamily domain can be searched for from those defined in sequence databases, for example, as a protein having a domain defined as "GH3 superfamily" on the NCBI Conserved domains database.

[0017] GH3 proteins, particularly those derived from plants, can be classified into groups I, II, and III based on sequence similarity and substrate specificity (J. Biol. Chem., 2010, 285, 29780-29786; Plant Cell., 2005, 17(2). 616-627).

[0018] Group I is a group of enzymes that were found to use jasmonic acid as a substrate. Examples of enzymes that belong to Group I include enzymes derived from Arabidopsis thaliana (e.g., AtGH3-10, AtJAR1 [also called AtGH3-11]), rice (Oryza sativa) (e.g., OsAK071721, OsBAA96221), tomato (Lycopersicon esculentum) (e.g., LeBTO13697, LeU144810), and Physcomitrella patens (PpABO61221).

[0019] Group II is a group of enzymes that have been found to use mainly indoleacetic acid or salicylic acid as substrates. Examples of enzymes belonging to group II include enzymes derived from Arabidopsis thaliana (e.g., AtGH3-1, AtGH3-2, AtGH3-3, AtGH3-4, AtGH3-5, AtGH3-6, AtGH3-9, AtGH3-17), enzymes derived from rice (Oryza sativa) (e.g., OsBAB63594, OsBAB92590, OsGH3-8 [also called OsBAC79627]), enzymes derived from pepper (Capsicum chinense) (e.g., CcAY525089), enzymes derived from soybean (Glycine max) (e.g., GmGH3), enzymes derived from tomato (Lycopersicon esculentum) (e.g., LeBT013446), and enzymes derived from tobacco (Nicotiana tabacum) (e.g., NtAF123503).

[0020] Group III is a group of enzymes that have been found to use mainly substituted benzoates as substrates. Examples of enzymes that belong to Group III include enzymes derived from Arabidopsis thaliana (e.g., AtGH3-7, AtGH3-8, AtGH3-12, AtGH3-13, AtGH3-14, AtGH3-15, AtGH3-16, AtGH3-18, and AtGH3-19).

[0021] Among GH3 proteins, examples of GH3 proteins derived from microorganisms include enzymes derived from Cystobacter fuscus (e.g., CfHP [WP_002626336]) and enzymes derived from Synechococcus sp. (e.g., SsGH3 [GH3 auxin-responsive promoter superfamily]).

[0022] The GH3 protein may be: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, which contains one or several amino acid substitutions, deletions, insertions, or additions, and which has N-acylase activity; or (C) A protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and having N-acylase activity.

[0023] The enzyme used in the method of the present invention may be a PaaK protein. The term "PaaK protein" refers to a group of enzymes and their homologs that have the function of converting phenylacetate to phenylacetate-CoA. As a structural feature, the term "PaaK protein" refers to a group of enzymes that contain a PaaK superfamily domain. The PaaK superfamily domain can be searched from those defined in a sequence database, for example, as a protein having a domain defined as "PaaK superfamily" on the NCBI Conserved domains database. The PaaK protein may also be found in a sequence database as a homolog of the GH3 protein, and may have, for example, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more of amino acid sequence identity with the GH3 protein.

[0024] Examples of PaaK proteins include indoleacetic acid-lysine synthetase (IAAL), which binds lysine to indoleacetic acid, such as an enzyme derived from Pseudomonas savastanoi (e.g., PsIAAL) and an enzyme derived from Pantoea agglomerans (e.g., PaHP [WP_031591948]).

[0025] The PaaK protein may be: (A') a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11; (B') a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 10 or 11 and having N-acylase activity; or (C') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 or 11, and having N-acylase activity.

[0026] In proteins (B) and (B'), one or several amino acid residues can be modified by one, two, three or four types of mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. Mutations of amino acid residues may be introduced into one region in the amino acid sequence, or into several different regions. The term "one or several" refers to a number that does not significantly impair the activity of the protein. The number referred to by the term "one or several" is, for example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, and particularly preferably 1 to 10 or 1 to 5 (e.g., 1, 2, 3, 4 or 5).

[0027] In the case of proteins (C) and (C'), the percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9 or the amino acid sequence of SEQ ID NO: 10 or 11 is 90% or more. Preferably, the identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The percent identity of a polypeptide (protein) can be calculated using the algorithm blastp. More specifically, the percent identity of a polypeptide can be calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11 Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) in the algorithm blastp provided by the National Center for Biotechnology Information (NCBI). The percent identity of a polynucleotide (gene) can be calculated using the algorithm blastn. More specifically, the percent identity of polynucleotides can be calculated using the algorithm blastn provided by NCBI with default Scoring Parameters (Match / Mismatch Scores=1, -2; Gap Costs=Linear).

[0028] "N-acylase activity" refers to the activity of producing an N-acyl-amino group-containing compound using an amino group-containing compound and a carboxyl group-containing compound as substrates. As for the GH3 protein and the PaaK protein, the proteins (A) to (C) and (A') to (C') have N-acylase activity, and therefore can produce an N-acyl-amino group-containing compound from an amino group-containing compound and a carboxyl group-containing compound. When the activity of each of the proteins (B), (B'), (C), and (C') is measured under specific measurement conditions, the activity of the protein (A) or (A') corresponding to the original amino acid sequence may be, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, particularly preferably 90% or more, 94% or more, 96% or more, 98% or more, or the same (i.e., 100%) or more. As such specific measurement conditions, the following conditions can be adopted. Protein (A) or (A') (hereinafter referred to as "wild-type enzyme") and protein (B), (B'), (C), or (C') (hereinafter referred to as "modified enzyme") were prepared as purified enzymes and incubated in 50 mM Tris-HCl, 5 mM amino acids (e.g., glycine, L-glutamic acid, L-aspartic acid), 5 mM sodium fatty acid (e.g., sodium caprylate, sodium caprate, sodium laurate), 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 50 μg / mL Incubate 0.2 mL of the purified enzyme, pH 8.0, reaction solution at 25°C for 24 hours. After the reaction is completed, add 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% methanol). Filter the mixture and subject it to UPLC-MS analysis to evaluate N-acylase activity by measuring the signal of the molecular weight corresponding to N-acyl amino acid (e.g., Nα-capryloyl glycine, Nα-caprinoyl glycine, Nα-lauroyl glycine, Nα-capryloyl-L-glutamic acid, Nα-caprinoyl-L-glutamic acid, Nα-lauroyl-L-glutamic acid, Nα-capryloyl-L-aspartic acid, Nα-caprinoyl-L-aspartic acid, Nα-lauroyl-L-aspartic acid).

[0029] Proteins (B), (B'), (C), and (C') may have mutations introduced into the catalytic domain and into the non-catalytic domain as long as the desired properties are retained. The positions of amino acid residues that may be mutated to retain the desired properties are clear to those skilled in the art. Specifically, those skilled in the art can 1) compare the amino acid sequences of multiple proteins with similar properties, 2) identify relatively conserved regions and relatively non-conserved regions, and then 3) predict regions that may play an important role in function and regions that may not play an important role in function from the relatively conserved regions and relatively non-conserved regions, respectively, and thus recognize the correlation between structure and function. Thus, those skilled in the art can identify the positions of amino acid residues that may be mutated in the amino acid sequence of the protein used in the present invention.

[0030] When an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution. As used herein, the term "conservative substitution" refers to replacing a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Preferably, conservative amino acid substitutions may be between aspartic acid and glutamic acid, between arginine, lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0031] The protein used in the present invention may also be a fusion protein linked to a heterologous moiety via a peptide bond. Examples of such heterologous moieties include peptide moieties that facilitate purification of the target protein (e.g., tag moieties such as histidine tag and Strep-tag II; proteins used for purification of the target protein such as glutathione-S-transferase, maltose-binding protein, and mutants thereof), peptide moieties that improve the solubility of the target protein (e.g., Nus-tag), peptide moieties that act as chaperones (e.g., trigger factor), peptide moieties with other functions (e.g., full-length proteins or parts thereof), and linkers.

[0032] The amino group-containing compound that can be used in the method of the present invention may be either an organic compound containing an amino group in which the nitrogen atom is bonded to one or two hydrogen atoms, or an organic compound containing an amino group in which the nitrogen atom is not bonded to a hydrogen atom. From the viewpoint of the substrate specificity of the enzyme, the amino group-containing compound is preferably a compound containing an amino group in which the nitrogen atom is bonded to one or two hydrogen atoms, and more preferably a compound containing an amino group in which the nitrogen atom is bonded to two hydrogen atoms.

[0033] The amino group-containing compound that can be used in the method of the present invention is preferably an amino group-containing compound having an anionic group, such as a carboxyl group, a sulfonic acid group, a sulfate group, or a phosphate group.

[0034] Examples of amino group-containing compounds having a carboxyl group as an anionic group include amino acids and peptides.

[0035] Examples of amino acids include α-amino acids, β-amino acids, and γ-amino acids. Examples of α-amino acids include glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine. Examples of β-amino acids include β-alanine. Examples of γ-amino acids include γ-aminobutyric acid. The amino group of an amino acid may be any of an amino group in which a nitrogen atom is bound to two hydrogen atoms, an amino group in which a nitrogen atom is bound to one hydrogen atom, and an amino group in which a nitrogen atom is not bound to a hydrogen atom. Examples of amino acids containing an amino group in which a nitrogen atom is bound to one hydrogen atom include sarcosine, N-methyl-β-alanine, N-methyltaurine, and proline. The amino acid may be any of L-amino acids and D-amino acids.

[0036] A peptide is a compound having a structure in which the above-mentioned amino acids are linked by amide bonds. Examples of peptides include oligopeptides (e.g., dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, and octapeptides) having a structure in which 2 to 10 amino acids are linked by amide bonds, and polypeptides (proteins) having a structure in which 11 or more amino acids are linked by amide bonds. Examples of dipeptides include aspartylphenylalanine, glycylglycine, β-alanylhistidine, and alanylglutamine.

[0037] Examples of amino group-containing compounds having a sulfonic acid group as an anionic group include taurine, N-methyltaurine, and cysteic acid.

[0038] Examples of amino group-containing compounds having a sulfate group as an anionic group include O-sulfoserine and O-sulfothreonine.

[0039] Examples of amino group-containing compounds having a phosphate group as an anionic group include ethanolamine phosphate, phosphoserine, and phosphothreonine.

[0040] The carboxyl group-containing compound that can be used in the method of the present invention is a compound that contains an unsubstituted carboxyl group (e.g., free form, ion, salt). Examples of the carboxyl group-containing compound include fatty acids, aromatic carboxylic acids, and indole carboxylic acids.

[0041] The fatty acid may be, for example, a fatty acid having 6 to 18 carbon atoms, preferably a fatty acid having 6 to 16 carbon atoms, more preferably a fatty acid having 6 to 14 carbon atoms, and even more preferably a fatty acid having 6 to 12 carbon atoms. Examples of fatty acids having 6 to 18 carbon atoms include caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonic acid (C9), capric acid (C10), undecylic acid (C11), lauric acid (C12), tridecylic acid (C13), myristic acid (C14), pentadecylic acid (C15), palmitic acid, palmitoleic acid, sapienic acid (all C16), margaric acid (C17), stearic acid, α-linolenic acid, γ-linolenic acid, linoleic acid, vaccenic acid, and oleic acid (all C18) (the number in parentheses indicates the number of carbon atoms). In addition, mixed fatty acids such as coconut oil fatty acid, palm fatty acid, and hardened beef tallow fatty acid can also be used.

[0042] The fatty acid is preferably a saturated fatty acid. Examples of the saturated fatty acids among the above fatty acids include caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, and stearic acid.

[0043] Examples of aromatic carboxylic acids include benzoic acid, phthalic acid, isophthalic acid, terephthalic acid, salicylic acid, gallic acid, and cinnamic acid.

[0044] The N-acyl-amino group-containing compound produced by the method of the present invention is a compound having a structure in which the amino group of the amino group-containing compound and the carboxyl group of the carboxyl group-containing compound form an amide bond. The N-acyl-amino group-containing compound is produced by the reaction of the amino group-containing compound and the carboxyl group-containing compound in the presence of the enzyme. The amino group that reacts with the carboxyl group may be at any position of the amino group-containing compound, for example, any of the α-position, β-position, γ-position, δ-position, and ε-position.

[0045] The enzymes used in the method of the present invention can be natural or recombinant proteins. Recombinant proteins can be obtained, for example, using cell-free vectors or from microorganisms that produce the enzymes used in the present invention. The enzymes used in the present invention can be used as unpurified, crude or purified enzymes. These enzymes may be used as immobilized proteins immobilized on a solid phase in the reaction.

[0046] The enzyme used in the method of the present invention is isolated by a known method, and further purified as necessary to obtain the desired enzyme. From the viewpoint of obtaining a large amount of the enzyme, transformed microorganisms are preferable as the microorganisms that produce the enzyme. In the present invention, the term "transformation" refers not only to the introduction of a polynucleotide into a host cell, but also to the modification of the genome in the host cell.

[0047] The culture conditions for the transformed microorganism are not particularly limited, and standard cell culture conditions can be used depending on the host. Media for culturing the transformed microorganism are known, and for example, nutrient media such as LB medium or minimal media such as M9 medium can be used by adding a carbon source, a nitrogen source, a vitamin source, and the like. The culture temperature is preferably 4 to 40°C, more preferably 10 to 37°C. The culture time is preferably 5 to 168 hours, more preferably 8 to 72 hours. The gas composition preferably has a CO2 concentration of about 6% to about 84%, and a pH of about 5 to 9. In addition, culture is preferably performed under aerobic, anaerobic, or anaerobic conditions depending on the properties of the host cells.

[0048] Any suitable method can be used as the culture method. Depending on the host cell, either shaking culture or stationary culture is possible, but stirring or aeration may be performed as necessary. Examples of such culture methods include batch culture, fed-batch culture, and continuous culture. When the expression of a specific protein produced by a transformed microorganism is under the control of an inducible promoter such as the lac promoter, the expression of the protein may be induced by adding an inducer such as IPTG (isopropyl-β-thiogalactopyranoside) to the culture medium.

[0049] The produced target enzyme can be purified and isolated from the extract of the transformed microorganism by known precipitation methods such as salting out, isoelectric precipitation or solvent precipitation, methods utilizing molecular weight differences such as dialysis, ultrafiltration or gel filtration, methods utilizing specific affinity such as ion exchange chromatography, methods utilizing differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, other affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing, or a combination of these. When the target enzyme is secreted and expressed, the culture supernatant containing the target enzyme can be obtained by removing the bacterial cells from the culture solution obtained by culturing the transformed microorganism by centrifugation or the like. The target enzyme can also be purified and isolated from this culture supernatant.

[0050] The reaction in the presence of the enzyme may be carried out using a transformed microorganism that produces the enzyme or a processed product thereof (eg, disrupted, lysed or lyophilized product of the microorganism).

[0051] Preferably, the polynucleotide encoding the enzyme used in the present invention may be a polynucleotide selected from the group consisting of the following (a) to (d): (a) a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12 to 22; (b) a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12 to 22 and encodes a protein having N-acylase activity; (c) a polynucleotide comprising a nucleotide sequence having 90% or more identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12 to 22 and encoding a protein having N-acylase activity; and (d) A degenerate variant of a polynucleotide selected from the group consisting of (a) to (c).

[0052] The polynucleotide may be either DNA or RNA, but is preferably DNA. The base sequences of SEQ ID NOs: 12 to 22 code for the amino acid sequences of SEQ ID NOs: 1 to 11, respectively.

[0053] In the above polynucleotide (b), the term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, stringent conditions include hybridization in 6×SSC (sodium chloride / sodium citrate) at about 45° C., followed by one or more washings in 0.2×SSC, 0.1% SDS at 50 to 65° C.

[0054] In the above polynucleotide (c), the percent identity of the base sequence to the base sequences of SEQ ID NOs: 12 to 22 may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0055] In the above polynucleotide (d), the term "degenerate mutant" refers to a polynucleotide mutant in which at least one codon encoding a specific amino acid residue in the polynucleotide before mutation is changed to another codon encoding the same amino acid residue. Since such a degenerate mutant is a mutant based on silent mutation, the protein (enzyme) encoded by the degenerate mutant is identical to the protein (enzyme) encoded by the polynucleotide before mutation.

[0056] Preferably, the degenerate variant is a polynucleotide variant in which the codons are altered to match the codon usage of the host cell into which it is to be introduced. When a gene is expressed in a heterologous host cell (e.g., a microorganism), differences in codon usage may result in insufficient supply of the corresponding tRNA molecule species, resulting in reduced translation efficiency and / or inaccurate translation (e.g., translation termination). For example, in Escherichia coli, the low-frequency codons shown in Table 1 are known.

[0057] [Table 1]

[0058] Therefore, in the present invention, a degenerate mutant that is compatible with the codon usage frequency of a host cell as described below can be used. For example, a degenerate mutant may have a codon that encodes one or more amino acid residues selected from the group consisting of arginine residues, glycine residues, isoleucine residues, leucine residues, and proline residues altered. More specifically, a degenerate mutant may have one or more codons that are low in frequency (e.g., AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC) altered. Preferably, a degenerate mutant may include alterations in one or more (e.g., one, two, three, four, or five) codons selected from the group consisting of the following: i) changing at least one codon selected from the group consisting of the four codons encoding Arg (AGG, AGA, CGG, and CGA) to another codon encoding Arg (CGU, or CGC); ii) changing one codon encoding Gly (GGA) to another codon (GGG, GGU, or GGC); iii) changing one codon (AUA) encoding Ile to another codon (AUU, or AUC); iv) changing one codon (CUA) encoding Leu to another codon (UUG, UUA, CUG, CUU, or CUC); and v) Changing one codon (CCC) that encodes Pro to another codon (CCG, CCA, or CCU). When the degenerate mutant is RNA, the nucleotide residue "U" should be utilized as described above, whereas when the degenerate mutant is DNA, the nucleotide residue "T" should be utilized instead of "U." The number of nucleotide residue mutations to match the codon usage frequency of the host cell is not particularly limited as long as the same protein is encoded before and after the mutation, and is, for example, 1 to 400, 1 to 300, 1 to 200, or 1 to 100.

[0059] Identification of low-frequency codons can be easily performed based on the type of any host cell and genome sequence information by utilizing techniques known in the art. Thus, degenerate mutants may include changes of low-frequency codons to non-low-frequency codons (e.g., high-frequency codons). In addition, since methods for designing mutants taking into consideration factors such as compatibility with the genomic GC content of the production strain as well as low-frequency codons are known (Alan Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments, BMC Bioinformatics. 2006 Jun 6;7:285.), such methods may be used. In this way, the above-mentioned mutants can be appropriately prepared depending on the type of any host cell (e.g., a microorganism as described below) into which they can be introduced.

[0060] The transformed microorganism in which the activity of the above enzyme is improved as compared to a wild-type microorganism is preferably a microorganism containing an expression unit comprising a polynucleotide encoding the above enzyme and a promoter operably linked thereto.

[0061] In the present invention, the term "expression unit" refers to a minimum unit that includes a predetermined polynucleotide to be expressed as a protein and a promoter operably linked thereto, and enables the transcription of the polynucleotide and thus the production of the protein encoded by the polynucleotide. The expression unit may further include elements such as a terminator, a ribosome binding site, and a drug resistance gene. The expression unit may be DNA or RNA, but is preferably DNA. The expression unit may also be homologous (i.e., inherent) or heterologous (i.e., non-inherited) to the host cell. The expression unit may also be an expression unit that includes one polynucleotide to be expressed as a protein and a promoter operably linked thereto (i.e., an expression unit that allows the expression of monocistronic mRNA), or an expression unit that includes multiple polynucleotides to be expressed as proteins (e.g., 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and particularly preferably 10 or more polynucleotides) operably linked thereto (i.e., an expression unit that allows the expression of polycistronic mRNA). The expression unit can be contained in a genomic region (e.g., a natural genomic region that is a natural locus where the polynucleotide encoding the above-mentioned protein is inherently present, or a non-natural genomic region that is not the natural locus) or a non-genomic region (e.g., in the cytoplasm) in a microorganism (host cell). The expression unit may be contained in the genomic region at one or more (e.g., 1, 2, 3, 4, or 5) different positions. Specific forms of the expression unit contained in the non-genomic region include, for example, a plasmid, a virus vector, a phage, and an artificial chromosome.

[0062] The promoter constituting the expression unit is not particularly limited as long as it can express in a host cell a protein (enzyme) encoded by a polynucleotide linked downstream of the promoter. For example, the promoter may be homologous or heterologous to the host cell. For example, a constitutive or inducible promoter that is commonly used for the production of recombinant proteins can be used. Examples of such promoters include PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, and tetracycline-inducible promoter. Preferably, a promoter having a strong transcription activity in a host cell can be used. Examples of promoters having a strong transcription activity in a host cell include promoters of genes highly expressed in the host cell and virus-derived promoters.

[0063] In one embodiment, the transformed microorganism in which the activity of the enzyme is improved compared to a wild-type microorganism may be a microorganism (i) comprising a heterologous expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto. The term "heterologous expression unit" means that the expression unit is heterologous to the host cell. Thus, in the present invention, at least one element constituting the expression unit is heterologous to the host cell. Examples of elements constituting the expression unit that are heterologous to the host cell include the elements described above. Preferably, one or both of the polynucleotide encoding the target enzyme or the promoter constituting the heterologous expression unit are heterologous to the host cell. Thus, in the present invention, one or both of the polynucleotide encoding the target enzyme or the promoter are derived from an organism other than the host cell (e.g., prokaryotes and eukaryotes, or animals such as microorganisms, insects, plants, and mammals) or viruses, or are artificially synthesized. As the heterologous expression unit, a heterologous expression unit in which at least one element constituting the expression unit is heterologous to the host cell is preferred.

[0064] In the microorganism (i), the protein constituting the expression unit may be heterologous to the host cell. Examples of such a microorganism include a microorganism comprising an expression unit comprising a polynucleotide encoding any one of the following proteins (A'') to (C'') and a promoter operably linked thereto: (A'') a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11; (B'') a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, which contains one or several amino acid substitutions, deletions, insertions, or additions, and which has N-acylase activity; or (C'') A protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, and having N-acylase activity.

[0065] In another embodiment, the transformed microorganism in which the activity of the enzyme is improved compared to a wild-type microorganism may be (ii) a microorganism comprising an expression unit in a non-native genomic region or in a non-genomic region, the expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto.

[0066] In yet another embodiment, the transformed microorganism in which the activity of the enzyme is improved compared to that of a wild-type microorganism may be (iii) a microorganism containing a polynucleotide encoding the enzyme in an expression unit in a plurality of copies, for example, 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and particularly preferably 10 or more copies.

[0067] In yet another embodiment, the transformed microorganism in which the activity of the enzyme is improved compared to a wild-type microorganism may be (iv) a microorganism containing a non-natural expression unit in which a mutation has been introduced into a unique expression unit (e.g., a promoter region) so as to enhance expression of the enzyme, or (v) a microorganism containing a non-natural expression unit in which a mutation has been introduced into a polynucleotide encoding the enzyme by a technique such as genome editing so as to improve the activity of the enzyme.

[0068] Preferably, the transformed microorganism in which the activity of the enzyme is improved as compared to that of a wild-type microorganism is any one of the microorganisms (i) to (iii).

[0069] In the present invention, host cells used as transformed microorganisms include bacteria, such as bacteria belonging to the Enterobacteriaceae family, and fungi. The bacteria may be gram-positive or gram-negative. Examples of gram-positive bacteria include bacteria of the genus Bacillus and bacteria of the genus Corynebacterium. As the bacteria of the genus Bacillus, Bacillus subtilis is preferred. As the bacteria of the genus Corynebacterium, Corynebacterium glutamicum is preferred. Examples of gram-negative bacteria include bacteria of the genus Escherichia and bacteria of the genus Pantoea. As the bacteria of the genus Escherichia, Escherichia coli is preferred. As the Pantoea bacteria, Pantoea ananatis is preferable. As the fungus, microorganisms of the Saccharomyces and Schizosaccharomyces genera are preferable. As the Saccharomyces microorganism, Saccharomyces cerevisiae is preferable. As the Schizosaccharomyces microorganism, Schizosaccharomyces pombe is preferable.

[0070] In the present invention, the host cell used as the transformed microorganism may be, for example, a host in which the decomposition system of acylamino acids, fatty acids, or amino acids is weakened or defective. Examples of the host in which the decomposition system is weakened or defective include a host in which a protein such as an enzyme associated with the decomposition system is weakened or defective, and a host that produces an inhibitor of the protein such as an enzyme associated with the decomposition system. Examples of the host in which a protein such as an enzyme associated with the decomposition system is weakened or defective include a host containing a mutation in the host genome that reduces or deletes the expression level of the protein, and a host containing a mutation in the host genome that reduces or deletes the activity of the protein. Examples of the host that produces or enhances an inhibitor of a protein such as an enzyme associated with the decomposition system include a host into which an expression unit of the inhibitor is introduced by transformation, a host containing a mutation in the host genome that enhances the expression level of the inhibitor, and a host containing a mutation in the host genome that enhances the activity of the inhibitor. Examples of the protein such as an enzyme associated with the decomposition system of acylamino acids include acylase, and examples of the protein such as an enzyme associated with the decomposition system of fatty acids include acyl-CoA synthase.

[0071] In the present invention, the host cell used as the transformed microorganism may be, for example, a host with enhanced ability to uptake amino acids and fatty acids in order to improve the efficiency of supplying substrates for the enzyme reaction and thereby improve production efficiency. Examples of the host with enhanced uptake ability include a host that produces or enhances a protein such as an enzyme related to the uptake ability. Examples of the host that produces or enhances a protein such as an enzyme related to the uptake ability include a host into which an expression unit of the protein has been introduced by transformation, a host containing a mutation in the host genome that enhances the expression amount of the protein, and a host containing a mutation in the host genome that enhances the activity of the protein.

[0072] The transformed microorganism used in the present invention can be prepared by any method known in the art. For example, the transformed microorganism as described above can be prepared by a method using an expression vector (e.g., competent cell method, electroporation method) or a genome modification technique. When the expression vector is an integrative vector that undergoes homologous recombination with the genomic DNA of the host cell, the expression unit can be integrated into the genomic DNA of the host cell by transformation. On the other hand, when the expression vector is a non-integrative vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit is not integrated into the genomic DNA of the host cell by transformation, and can exist in the host cell as an expression vector, independent of the genomic DNA. Alternatively, according to genome editing techniques (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)), it is possible to integrate the expression unit into the genomic DNA of the host cell and to modify the expression unit inherent to the host cell.

[0073] In addition to the above-mentioned minimal expression unit, the expression vector may further include elements that function in a host cell, such as a terminator, a ribosome binding site, and a drug resistance gene, for example, resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0074] The expression vector may further include a region that allows homologous recombination with the genome of the host cell for homologous recombination with the genomic DNA of the host cell. For example, the expression vector may be designed so that the expression unit contained therein is located between a pair of homologous regions (e.g., homology arms that are homologous to a specific sequence in the genome of the host cell, loxP, FRT). The genomic region of the host cell into which the expression unit is to be introduced (target of the homologous region) is not particularly limited, and may be the locus of a gene that is highly expressed in the host cell.

[0075] The expression vector may be a plasmid, a virus vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. The integrative vector may be a type of vector that is integrated into the genome of a host cell in its entirety. Alternatively, the integrative vector may be a type of vector that is integrated into the genome of a host cell only in part (e.g., an expression unit). The expression vector may further be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), pET (e.g., pET28a), and derivatives thereof.

[0076] The amino group-containing compound and carboxyl group-containing compound, which are substrates used in the method of the present invention, can be added to a reaction system containing the enzyme (e.g., an aqueous solution containing the enzyme, a culture solution containing a transformed microorganism that produces the enzyme, or a processed product of the transformed microorganism that produces the enzyme). Alternatively, in the method of the present invention, an amino group-containing compound or a carboxyl group-containing compound produced in a separate reaction system can be used as a substrate.

[0077] When the method of the present invention is carried out using the enzyme itself (e.g., purified enzyme), an aqueous solution containing the enzyme can be used as the reaction system. A buffer solution is preferable as the aqueous solution. Examples of the buffer solution include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer. The pH is preferably about 5 to 10, for example. The amounts of the enzyme, amino group-containing compound, and carboxyl group-containing compound (substrate) in the reaction system, and the reaction time can be appropriately adjusted depending on the amount of N-acyl-amino group-containing compound to be produced. The reaction temperature is not particularly limited as long as the reaction proceeds, but 20 to 40°C is preferable.

[0078] The method of the present invention may be carried out in combination with an ATP regenerating system. When the method of the present invention is carried out using the enzyme itself (e.g., purified enzyme), the combination with the ATP regenerating system may be, for example, a reaction in combination with an ATP regenerating enzyme (e.g., a mixture). Examples of the ATP regenerating enzyme include polyphosphate kinase, a combination of polyphosphate:AMP phosphotransferase and polyphosphate kinase, and a combination of polyphosphate:AMP phosphotransferase and adenylate kinase. When the method of the present invention is carried out using a transformed microorganism that produces the enzyme or a processed product thereof, the combination with the ATP regenerating system may be, for example, a microorganism with enhanced ATP supplying ability used as a host. Examples of the microorganism with enhanced ATP supplying ability include the above-mentioned microorganisms that produce or enhance the ATP regenerating enzyme. Examples of the microorganism that produces or enhances the ATP regenerating enzyme include a host into which an expression unit of the ATP regenerating enzyme has been introduced by transformation, a host containing a mutation in the host genome that enhances the expression amount of the ATP regenerating enzyme, and a host containing a mutation in the host genome that enhances the activity of the ATP regenerating enzyme.

[0079] The production of the N-acyl-amino group-containing compound can be appropriately confirmed. For example, such confirmation can be performed by adding a reaction stop solution (e.g., 1% (v / v) phosphoric acid, 75% (v / v) aqueous methanol solution) to the reaction system, filtering the mixture, and then subjecting it to UPLC-MS analysis. EXAMPLES

[0080] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0081] Example 1: Expression and purification of acylamino acid synthetase 1) Construction of acylamino acid synthetase expression plasmid Arabidopsis thaliana-derived jasmonic acid-amido synthetase JAR1 (AtJAR1, Q9SKE2, SEQ ID NO: 3), Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.6 (AtGH3-6, Q9LSQ4, SEQ ID NO: 1), Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.5 (AtGH3-5, O81829, SEQ ID NO: 4), Arabidopsis thaliana-derived GH3-10 (AtGH3-10, OAO98077, SEQ ID NO: 5), Arabidopsis thaliana-derived 4-substituted benzoates-glutamate ligase GH3.12 (AtGH3-12, Q9LYU4, SEQ ID NO: 6), Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.17 (AtGH3-17, Q9FZ87, SEQ ID NO: 7), hypothetical protein derived from Cystobacter fuscus (CfHP, WP_002626336, SEQ ID NO: 9), GH3 auxin-responsive promoter superfamily derived from Synechococcus sp. PCC 7335 (SsGH3, WP_006458022, SEQ ID NO: 8), indoleacetate-lysine synthetase derived from Pseudomonas savastanoi (PsIAAL, P18204, SEQ ID NO: 10), hypothetical The gene encoding protein (PaHP, WP_031591948, SEQ ID NO: 11) was codon-optimized for expression in E. coli, and plasmid DNA inserted into the NdeI and XhoI sites in the multiple cloning site of pET-28a(+) (Merck) was purchased from Eurofins Genomics.The plasmids were named pET-28a-AtJAR1, pET-28a-AtGH3-6, pET-28a-AtGH3-5, pET-28a-AtGH3-10, pET-28a-AtGH3-12, pET-28a-AtGH3-17, pET-28a-CfHP, pET-28a-SsGH3, pET-28a-PsIAAL, and pET-28a-PaHP, respectively. These plasmids express proteins with a His-tag and a thrombin cleavage site fused to the N-terminus.

[0082] A synthetic DNA with codons optimized for expression in E. coli was purchased from GenScript for the gene encoding probable indole-3-acetic acid-amide synthetase GH3.8 (OsGH3-8, A3BLS0, SEQ ID NO: 2) derived from Oryza sativa. The synthetic DNA was treated with restriction enzymes NdeI and EcoRI, and ligated with pET28a(+) (Merck) similarly treated with NdeI and EcoRI. E. coli JM109 was transformed with this ligation solution, and the target plasmid was extracted from the kanamycin-resistant strain and named pET-28a-OsGH3-8. This plasmid expresses a protein with a His-tag and a thrombin cleavage site fused to the N-terminus.

[0083] 2) Expression of acylamino acid synthetase Plasmids pET-28a-AtJAR1, pET-28a-AtGH3-6, pET-28a-OsGH3-8, pET-28a-AtGH3-5, pET-28a-AtGH3-12, pET-28a-AtGH3-17, pET-28a-PsIAAL, and pET-28a-PaHP were introduced into E. coli BL21(DE3), and the transformants were inoculated into 100mL of LB containing 25mg / L kanamycin and cultured at 37℃ using a Sakaguchi flask. When OD610 reached 0.6, 1mM IPTG was added and cultured at 15℃ for 24 hours with shaking.

[0084] The plasmid pET-28a-CfHP was introduced into E. coli BL21(DE3), the transformant was inoculated into 100mL of LB containing 25mg / L kanamycin, and cultured with shaking at 37℃ using a Sakaguchi flask. When OD610 reached 0.2, 1mM IPTG was added, and cultured with shaking at 15℃ for 24 hours.

[0085] The plasmid pET-28a-AtGH3-10 was introduced into E. coli BL21(DE3), and the transformant was inoculated into 100 mL of TB containing 25 mg / L kanamycin, and cultured with shaking at 37°C using a Sakaguchi flask. When OD610 reached 0.4, 1 mM IPTG was added, and cultured with shaking at 15°C for 24 hours.

[0086] The plasmid pET-28a-SsGH3 was introduced into E. coli BL21(DE3), the transformant was inoculated into 100mL of TB containing 25mg / L kanamycin, and cultured with shaking at 37℃ using a Sakaguchi flask. When OD610 reached 0.2, 1mM IPTG was added, and cultured with shaking at 15℃ for 24 hours.

[0087] 3) Purification of acylamino acid synthetase After the cultivation, the cells were collected from the obtained culture medium by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0 or 10 mM imidazole, and then ultrasonically disrupted. The cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as the soluble fraction. The resulting soluble fraction was applied to a His-tag protein purification column His TALON superflow 5ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0 or 10 mM imidazole, and adsorbed to the carrier. Proteins that were not adsorbed to the carrier (non-adsorbed proteins) were washed away with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0 or 10 mM imidazole, and then adsorbed proteins were eluted at a flow rate of 5 mL / min with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 150 mM imidazole. The resulting fractions were collected and concentrated and buffer exchanged using 20 mM Tris-HCl (pH 8.0) and an Amicon Ultra-15 10 kDa (Merck). If necessary, the amount of culture medium was increased and purification was performed.

[0088] Example 2: Synthesis of N-caprinoyl amino acid using acylamino acid synthetase 0.2 mL of reaction solution containing 50 mM Tris-HCl, 5 mM amino acids, 5 mM sodium caprate, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 50 μg / mL purified enzyme, pH 8.0, was incubated for 24 hours at 25° C. After the reaction was completed, 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis, and a signal with a molecular weight consistent with N-caprinoyl amino acid was detected.

[0089] The UPLC-MS analysis conditions are as follows. Instrument: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7μm 2.1×100mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: Acetonitrile Gradient:

[0090] [Table 2]

[0091] Flow rate: 0.6mL / min Injection volume: 2μL Column temperature: 40℃ Ionization method: ESI-negative

[0092] As a result of UPLC-MS analysis, signals of molecular weights consistent with the corresponding N-caprinoyl amino acids were confirmed in reaction solutions in which the enzymes and amino acids shown in Table 3 below were combined.

[0093] [Table 3]

[0094] Example 3: Synthesis of N-caprinoyl-amino acid derivatives, N-caprinoyl-D-amino acids, and N-caprinoyl-peptides using acylamino acid synthetases 0.2 mL of reaction solution containing 50 mM Tris-HCl, 5 mM amino acid derivatives, or D-amino acids, or peptides, 5 mM sodium caprate, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 50 μg / mL purified enzyme, pH 8.0, was incubated at 25° C. for 24 hours. After the reaction was completed, 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis to detect signals of molecular weights corresponding to N-caprinoyl amino acid derivatives, N-caprinoyl-D-amino acids, or N-caprinoyl-peptides. The UPLC-MS analysis conditions were as described in Example 2.

[0095] As a result of UPLC-MS analysis, in the reaction solution in which the enzymes shown in Table 4 below were combined with the amino acid derivatives, D-amino acids, or peptides, signals of molecular weights corresponding to the corresponding N-caprinoyl-amino acid derivatives, N-caprinoyl-D-amino acids, or N-caprinoyl-peptides were confirmed.

[0096] [Table 4]

[0097] Example 4: Synthesis of N-lauroylamino acid and N-lauroyl-amino acid derivatives using acylamino acid synthetase A reaction solution of 50 mM Tris-HCl, 5 mM amino acid or amino acid derivative, 5 mM sodium laurate, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 200 μg / mL purified enzyme, pH 8.0, 0.2 mL was incubated at 25 ° C. for 24 hours. After the reaction was completed, 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis, and signals of molecular weights corresponding to N-lauroyl amino acid and N-lauroyl-amino acid derivative were detected. The UPLC-MS analysis conditions were as described in Example 2.

[0098] As a result of UPLC-MS analysis, signals of molecular weights corresponding to the corresponding N-lauroyl amino acids and N-lauroyl-amino acid derivatives were confirmed in reaction solutions in which the enzymes and amino acids or amino acid derivatives shown in Table 5 below were combined.

[0099] [Table 5]

[0100] Example 5: Synthesis of N-acylamino acids using acylamino acid synthetase 0.1 mL of reaction solution containing 50 mM Tris-HCl, 5 mM amino acid, 5 mM fatty acid sodium salt, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 200 μg / mL purified enzyme, pH 8.0, was incubated at 25 ° C for 24 hours. L-Asp was used for AtGH3-6, OsGH3-8, AtGH3-5, and AtGH3-12, and Gly or L-Ala was used for CfHP. After the reaction was completed, 0.4 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis, and the N-acyl amino acid produced was quantified by UV210 nm detection. The UPLC-MS analysis conditions were as described in Example 2. The analysis results showed that when AtGH3-6 was used, Nα-capryloyl-L-aspartic acid was 3.9 mM, Nα-caprinoyl-L-aspartic acid was 4.5 mM, and Nα-lauroyl-L-aspartic acid was 2.2 mM. When OsGH3-8 was used, Nα-capryloyl-L-aspartic acid was 4.3 mM, Nα-caprinoyl-L-aspartic acid was 4.6 mM, and Nα-lauroyl-L-aspartic acid was 3.5 mM. When AtGH3-5 was used, Nα-capryloyl-L-aspartic acid was 4.1 mM, Nα-caprinoyl-L-aspartic acid was 4.6 mM, and Nα- The concentrations of lauroyl-L-aspartic acid were 2.5 mM. When AtGH3-12 was used, the concentrations of Nα-capryloyl-L-aspartic acid were 1.6 mM, Nα-caprinoyl-L-aspartic acid were 0.6 mM, and Nα-lauroyl-L-aspartic acid were 0.2 mM. When CfHP was used, the concentrations of Nα-capryloylglycine were 4.5 mM, Nα-caprinoylglycine was 4.6 mM, Nα-lauroylglycine was 0.1 mM, Nα-capryloyl-L-alanine was 3.1 mM, Nα-caprinoyl-L-alanine was 3.6 mM, and Nα-lauroyl-L-alanine was 0.4 mM.

[0101] Example 6: Synthesis of N-acylamino acids using acylamino acid synthetase The reaction mixture was 50 mM Tris-HCl, 5 mM amino acid, 5 mM fatty acid sodium salt (sodium palmitate and sodium stearate were 3 mM), 10 mM ATP, 10 mM MgCl2, 1 mM DTT, 200 μg / mL purified enzyme, pH 8.0, and 0.1 mL of the reaction mixture was shaken at 25 °C for 24 hours. When sodium palmitate or sodium stearate was used, the reaction mixture contained methanol at a final concentration of 10% (v / v). The amino acids used were L-Asp for AtGH3-6, OsGH3-8, AtGH3-5, and AtGH3-12, L-Ile for AtJAR1, L-Ala for AtGH3-10 and SsGH3, L-Glu for AtGH3-17, Gly for CfHP, L-Lys for PsIAAL, and L-Cys for PaHP. After the reaction was completed, 0.4 mL of a reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and then subjected to UPLC-MS analysis to detect a signal with a molecular weight consistent with that of an N-acyl amino acid.

[0102] The UPLC-MS analysis conditions are as follows. Instrument: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 × 100 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: Acetonitrile Gradient:

[0103] [Table 6]

[0104] Flow rate: 0.6mL / min Injection volume: 2μL Column temperature: 40℃ Ionization method: ESI-negative

[0105] As a result of UPLC-MS analysis, signals of molecular weights corresponding to the corresponding N-acyl amino acids were confirmed in the reaction solutions in which the enzymes shown in Table 7 below were combined with sodium fatty acids.

[0106] [Table 7]

[0107] Example 7: Analysis of ATP dependence of acylamino acid synthetase 0.25 mL of reaction solution containing 50 mM Tris-HCl, 5 mM amino acid, 5 mM sodium caprate, 10 mM or 0 mM ATP, 10 mM MgCl2, 1 mM DTT, 50 μg / mL purified enzyme, pH 8.0, was incubated at 25 ° C for 24 hours. L-Asp was used for AtGH3-6, and Gly was used for CfHP. After the reaction was completed, 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added to 0.2 mL of reaction solution, and the mixture was subjected to UPLC-MS analysis after filtration and the N-caprinoyl amino acid produced was quantified by UV210 nm detection. The UPLC-MS analysis conditions were as described in Example 2. As a result of the analysis, in the presence of ATP, 3.8 mM N-caprinoyl amino acid was detected in AtGH3-6 and 5.2 mM in CfHP, but in the absence of ATP, no signal of molecular weight corresponding to N-caprinoyl amino acid was detected in either AtGH3-6 or CfHP.

[0108] Example 8: Synthesis of N-capryloylamino acid using acylamino acid synthetase-expressing bacteria (1) Preparation of various bacterial cell liquids BL21(DE3) / pET-28a-AtGH3-6, BL21(DE3) / pET-28a-OsGH3-8, BL21(DE3) / pET-28a-AtGH3-5, and BL21(DE3) / pET-28a were inoculated into 100 mL of LB containing 25 mg / L kanamycin, and cultured with shaking at 37°C using a Sakaguchi flask. When OD610 reached 0.6, 1 mM IPTG was added, and cultured with shaking at 15°C for 24 hours. BL21(DE3) / pET-28a-CfHP was inoculated into 100 mL of LB containing 25 mg / L kanamycin, and cultured with shaking at 37°C using a Sakaguchi flask. When OD610 reached 0.2, 1 mM IPTG was added, and cultured with shaking at 15°C for 24 hours.

[0109] (Preparation of cell-free extract) After the cultivation, the cells were collected from 5 mL of the obtained culture medium by centrifugation, washed with 20 mM Tris-HCl (pH 7.6), and then suspended in 1 mL of BugBuster (registered trademark) Master Mix (Merck). After incubation at room temperature for 10 to 20 minutes, the supernatant was collected by centrifugation and used as a cell-free extract. (Preparation of washed bacterial fluid) After the cultivation was completed, the cells were collected from 5 mL of the obtained culture medium by centrifugation, washed with 20 mM Tris-HCl (pH 7.6), and then suspended in 1 mL of 20 mM Tris-HCl (pH 7.6) to prepare a washed cell liquid. (Preparation of bacterial cell suspension) After the cultivation was completed, 15 mL of the obtained culture solution was concentrated to 3 mL by centrifugation to obtain a bacterial cell solution.

[0110] (2) Synthesis of N-capryloyl amino acids using various bacterial cell fluids 0.3 mL of reaction solution containing 33.3 mM Tris-HCl, 3.3 mM amino acids, 3.3 mM sodium caprylate, 6.7 or 0 mM ATP, 6.7 mM MgCl2, 0.7 mM DTT, 30 μL of bacterial cell liquid (cell-free extract, washed bacterial cell liquid, bacterial cell liquid), pH 8.0, was incubated at 25 ° C for 24 hours. As amino acids, L-Asp was used for AtGH3-6, OsGH3-8, and AtGH3-5, and Gly or L-Ala was used for CfHP. After the reaction was completed, 0.8 mL of reaction stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added to 0.2 mL of the reaction solution, and the mixture was subjected to UPLC-MS analysis after filtration, and a signal with a molecular weight consistent with N-capryloyl amino acid was detected. The UPLC-MS analysis conditions were as described in Example 2. As a result of UPLC-MS analysis, signals of molecular weights consistent with the corresponding N-capryloyl amino acids were confirmed in the reaction solutions using the bacterial cell liquids and the like shown in Table 8 below.

[0111] [Table 8]

[0112] In the reaction using the purified enzyme, no N-acylamino acids were produced in the absence of ATP (Example 7), but in the reaction using the bacterial cell fluid, N-acylamino acids were produced even in the absence of ATP, suggesting that the enzymatic reaction proceeded using ATP contained in the bacterial cells.

[0113] The N-acyl amino acids produced were quantified by UV detection at 210 nm. When Gly was used as a substrate, 2.8 mM N-capryloyl amino acids were detected in the washed bacterial cell fluid, 2.2 mM in the bacterial cell fluid (with ATP), and 2.6 mM in the bacterial cell fluid (without ATP). When L-Ala was used as a substrate, 2.4 mM N-capryloyl amino acids were detected in the washed bacterial cell fluid, 0.9 mM in the bacterial cell fluid (with ATP), and 1.0 mM in the bacterial cell fluid (without ATP). [Industrial Applicability]

[0114] INDUSTRIAL APPLICABILITY The present invention is useful for producing N-acyl-amino group-containing compounds that can be used as cosmetic ingredients (particularly, surfactants) and the like. [Sequence List Free Text]

[0115] SEQ ID NOs: 1 to 11 show the amino acid sequences of AtGH3-6, OsGH3-8, AtJAR1 (AtGH3-11), AtGH3-5, AtGH3-10, AtGH3-12, AtGH3-17, SsGH3, CfHP (WP_002626336), PsIAAL, and PaHP (WP_031591948), respectively. SEQ ID NOs: 12 to 22 show the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 1 to 11, respectively, in which the codons have been optimized for expression in Escherichia coli.

Claims

1. A method for producing an N-acyl-amino group-containing compound by reacting an amino group-containing compound with a carboxyl group-containing compound in the presence of a GH3 protein, The GH3 protein is (A) a protein comprising an amino acid sequence of SEQ ID NO: 2 to 8; (B) a protein comprising an amino acid sequence containing 1 or 50 amino acid substitutions, deletions, insertions, or additions in the amino acid sequences of SEQ ID NOs: 2 to 8, and having N-acylase activity; or (C) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequences of SEQ ID NOs: 2 to 8 and having N-acylase activity; Selected from the group consisting of: the amino group-containing compound is an amino acid or a peptide, or taurine; The carboxyl group-containing compound is a fatty acid having 6 to 16 carbon atoms. A method for producing an N-acyl-amino group-containing compound.

2. The method of claim 1, wherein the amino acid or peptide is an α-amino acid, β-amino acid, or γ-amino acid, or a dipeptide thereof.

3. 3. The method according to claim 1 or 2, wherein the amino acid is an L-amino acid or a D-amino acid.

4. The amino acid or peptide is selected from the group consisting of: (1) (a) an α-amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine; (b) β-alanine; (c) gamma-aminobutyric acid; and (d) sarcosine; an amino acid selected from the group consisting of: (2) a dipeptide selected from the group consisting of aspartylphenylalanine, glycylglycine, and alanylhistidine; The method according to any one of claims 1 to 3, wherein the compound is selected from the group consisting of:

5. The method described in any one of claims 1 to 4, wherein the fatty acid is a fatty acid having 6 to 12 carbon atoms.

6. A method described in any one of claims 1 to 5, wherein the fatty acid is a saturated fatty acid.

7. The method according to any one of claims 1 to 6, wherein the GH3 protein is a purified GH3 protein.

8. The method according to any one of claims 1 to 7, wherein the reaction in the presence of the GH3 protein is carried out using a transformed microorganism that produces the GH3 protein, or a processed product thereof.

9. The method according to claim 8, wherein the transformed microorganism is any one of the following microorganisms (i) to (iii): (i) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding the GH3 protein and a promoter operably linked thereto; (ii) a microorganism comprising an expression unit in a non-native genomic region or in a non-genomic region, the expression unit comprising a polynucleotide encoding the GH3 protein and a promoter operably linked thereto; or (iii) A microorganism comprising an expression unit containing a polynucleotide encoding the GH3 protein in multiple copies.

10. The method according to claim 8 or 9, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae.

11. The method of claim 10, wherein the bacterium is Escherichia coli.