Method for producing N-acyl-amino group-containing compounds

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). Chemical synthesis of N-acyl-amino group-containing compounds (e.g., Schotten-Baumann reaction) poses a problem of environmental impact due to by-products of the synthesis reaction. Therefore, enzymatic synthesis of N-acyl-amino group-containing compounds is desired. Several prior art techniques for the enzymatic synthesis of N-acyl-amino group-containing compounds have been reported.

[0003] Patent Document 1 reports the fermentation of Nα-acyl amino acids from sugars using the surfactin biosynthesis enzyme of Bacillus subtilis. However, the amount of Nα-acyl glutamic acid produced is only 116.8 mg / L, which is very small, making this fermentation unsuitable for industrial-scale production.

[0004] Patent Document 2 reports a method for synthesizing Nα-acylglycine from amino acids and fatty acids using a human amino acid N-acyltransferase and an E. coli acyl-CoA synthetase. However, this method cannot directly bind an amino acid to a fatty acid and requires a two-step enzymatic 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, this method is inefficient for industrial production because it requires the use of a large amount of glycerol and the synthesis of Nα-acylamino acids in an aqueous solvent containing no glycerol results in a low yield.

[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 that does not contain 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 discovered that an enzyme capable of linking a carboxyl group and an amino group to form an amide bond in an ATP-dependent manner can efficiently produce an N-acyl-amino group-containing compound from a carboxyl group-containing compound, including a fatty acid, and an amino group-containing compound, thereby completing 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 derived from a plant or a microorganism. [3] The method of [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 of Group I, Group II, or Group III. [5] The GH3 protein is one 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 having 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 [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: (A') a protein comprising the amino acid sequence of SEQ ID NO: 10 or 11; (B') a protein having an amino acid sequence of SEQ ID NO: 10 or 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 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 [1] to

[11] , 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 [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 containing 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 containing a polynucleotide encoding the enzyme in an expression unit 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. [Effects 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 INVENTION

[0013] The present invention provides a method for producing an N-acyl-amino group-containing compound, which 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 present invention have the ability to combine carboxyl and amino groups to form an amide bond in an ATP-dependent manner. It is believed that the enzymes used in the methods of the present invention activate a carboxyl-containing compound by adenylation, and then form an amide bond by a mechanism in which an amino-containing compound nucleophilically attacks this 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 the divisions Gymnosperms, Angiosperms, Pteridophytes, Lycopods, Hornworts, Bombyxophytes, Marchantiophytes, 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. "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, "GH3 protein" refers to a protein that contains a GH3 superfamily domain. GH3 superfamily domains can be searched for in sequence databases, for example, proteins having a domain defined as "GH3 superfamily" can be searched for in 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 discovered that primarily use jasmonic acid as a substrate. Examples of Group I enzymes 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 (Arabidopsis thaliana) (e.g., AtGH3-1, AtGH3-2, AtGH3-3, AtGH3-4, AtGH3-5, AtGH3-6, AtGH3-9, and AtGH3-17), enzymes derived from rice (Oryza sativa) (e.g., OsBAB63594, OsBAB92590, and OsGH3-8 [also referred to as OsBAC79627]), enzymes derived from chili 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 found to primarily use substituted benzoates as substrates, including, for example, Arabidopsis thaliana-derived enzymes (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 having 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. "PaaK protein" refers to a group of enzymes and their homologs that have the function of converting phenylacetic acid to phenylacetic acid-CoA. As a structural feature, "PaaK protein" refers to a group of enzymes that contain a PaaK superfamily domain. PaaK superfamily domains can be searched for in sequence databases, for example, proteins having a domain defined as "PaaK superfamily" in the NCBI Conserved Domains database. PaaK proteins may also be found as homologs of GH3 proteins in sequence databases, and may share, for example, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more amino acid sequence identity with GH3 proteins.

[0024] Examples of PaaK proteins include indoleacetic acid-lysine synthetase (IAAL), which binds lysine to indoleacetic acid, such as the enzyme derived from Pseudomonas savastanoi (e.g., PsIAAL) and the 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 of SEQ ID NO: 10 or 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 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. The amino acid residue mutations may be introduced into one region of 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 indicated 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] Proteins (C) and (C') have a percent identity of 90% or more 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 NOS: 10 or 11. 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. Calculation of percent identity of polypeptides (proteins) can be performed using the blastp algorithm. More specifically, calculation of percent identity of polypeptides can be performed using the blastp algorithm provided by the National Center for Biotechnology Information (NCBI) with default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). Calculation of percent identity of polynucleotides (genes) can be performed using the blastn algorithm. More specifically, the percent identity of polynucleotides can be calculated using the blastn algorithm provided by NCBI with default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0028] "N-acylase activity" refers to the activity of generating an N-acylamino group-containing compound using an amino group-containing compound and a carboxyl group-containing compound as substrates. Regarding GH3 protein and PaaK protein, proteins (A) to (C) and (A') to (C') possess N-acylase activity and are therefore capable of generating an N-acylamino group-containing compound from an amino group-containing compound and a carboxyl group-containing compound. When the activity of proteins (B), (B'), (C), and (C') is measured under specific measurement conditions, the activity 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 of the activity of protein (A) or (A') corresponding to the original amino acid sequence. The following specific measurement conditions can be used: 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 A 0.2 mL reaction mixture containing the purified enzyme, pH 8.0, is incubated at 25°C for 24 hours. After completion of the reaction, 0.8 mL of a reaction stop solution (1% (v / v) phosphoric acid, 75% methanol) is added. The mixture is filtered and then subjected to UPLC-MS analysis. N-acylase activity is assessed by measuring the signal corresponding to the molecular weight of N-acylamino acids (e.g., Nα-capryloylglycine, Nα-caprinoylglycine, Nα-lauroylglycine, 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, and Nα-lauroyl-L-aspartic acid).

[0029] Proteins (B), (B'), (C), and (C') may have mutations introduced into their catalytic domains or into other regions, as long as the desired properties are maintained. The positions of amino acid residues that can be mutated to maintain the desired properties are readily apparent to those skilled in the art. Specifically, a skilled artisan can 1) compare the amino acid sequences of multiple proteins with similar properties, 2) identify relatively conserved and relatively non-conserved regions, and then 3) predict regions that may play important roles in function and regions that may not play important roles in function from the relatively conserved and non-conserved regions, respectively, thereby recognizing the correlation between structure and function. Therefore, a skilled artisan can identify the positions of amino acid residues that can be mutated in the amino acid sequence of a 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 β-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 the purification of the target protein (e.g., tag moieties such as histidine tag and Strep-tag II; proteins used in the purification of target proteins such as glutathione S-transferase, maltose-binding protein, and their mutants), 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 portions 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 an amino group in which the nitrogen atom is bonded to two hydrogen atoms, an amino group in which the nitrogen atom is bonded to one hydrogen atom, or an amino group in which the nitrogen atom is not bonded to any hydrogen atom. Examples of amino acids containing an amino group in which the nitrogen atom is bonded to one hydrogen atom include sarcosine, N-methyl-β-alanine, N-methyltaurine, and proline. The amino acid may be either an L-amino acid or a D-amino acid.

[0036] Peptides are compounds 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., a free form, an ion, or a 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 numbers in parentheses indicate 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 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 reacting the amino group-containing compound with the carboxyl group-containing compound in the presence of the enzyme. The amino group that reacts with the carboxyl group may be located at any position of the amino group-containing compound, for example, at the α-, β-, γ-, δ-, or ε-position.

[0045] The enzymes used in the methods 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, partially purified, or purified enzymes. These enzymes may also be used as immobilized proteins immobilized on a solid phase in the reaction.

[0046] The enzyme used in the method of the present invention can be isolated by known methods and further purified as necessary to obtain the desired enzyme. From the viewpoint of obtaining large amounts of enzyme, transformed microorganisms are preferred 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 of 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 supplemented with a carbon source, nitrogen source, vitamin source, etc. can be used. 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. Furthermore, culture is preferably carried out under aerobic, anaerobic, or anaerobic conditions depending on the properties of the host cells.

[0048] Any appropriate culture method can be used. Depending on the host cell, either shaking culture or static culture is possible, with stirring or aeration being possible as necessary. Such culture methods include, for example, 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, protein expression 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 an 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, a 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 consisting of 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 encode 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 nonspecific hybrids are not formed. For example, stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at about 45°C, followed by one or more washes in 0.2xSSC, 0.1% SDS at 50-65°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 pre-mutation polynucleotide is changed to another codon encoding the same amino acid residue. Because 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 pre-mutation polynucleotide.

[0056] Preferably, a degenerate variant is a polynucleotide variant in which codons have been 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 an insufficient supply of corresponding tRNA molecular 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, the present invention can utilize degenerate mutants that are compatible with the codon usage frequency of the host cell, as described below. For example, a degenerate mutant may have an altered codon encoding one or more amino acid residues selected from the group consisting of arginine, glycine, isoleucine, leucine, and proline residues. More specifically, a degenerate mutant may have an altered codon selected from the group consisting of low-frequency codons (e.g., AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC). 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: i) changing at least one codon selected from the group consisting of 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 encoding Leu (CUA) to another codon (UUG, UUA, CUG, CUU, or CUC); and v) Changing one codon (CCC) encoding Pro to another codon (CCG, CCA, or CCU). When the degenerate mutant is RNA, the nucleotide residue "U" should be used as described above, but when the degenerate mutant is DNA, the nucleotide residue "T" should be used instead of "U." The number of nucleotide residues mutated 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 may be, 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 host cell and genome sequence information using techniques known in the art. Therefore, degenerate mutants may contain changes from low-frequency codons to non-low-frequency codons (e.g., high-frequency codons). Furthermore, methods for designing mutants that take into account 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.), and such methods may also be used. Thus, the above-mentioned mutants can be appropriately prepared depending on the type of 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 enzyme is improved compared to that of a wild-type microorganism is preferably a microorganism containing an expression unit comprising a polynucleotide encoding the enzyme and a promoter operably linked thereto.

[0061] In the present invention, the term "expression unit" refers to the smallest unit that contains a polynucleotide to be expressed as a protein and a promoter operably linked thereto, enabling transcription of the polynucleotide and thus production of the protein encoded by the polynucleotide. The expression unit may further contain 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-native) to the host cell. The expression unit may also be an expression unit containing one polynucleotide to be expressed as a protein and a promoter operably linked thereto (i.e., an expression unit that enables monocistronic mRNA expression), or an expression unit containing multiple polynucleotides to be expressed as proteins (e.g., two or more, preferably three or more, more preferably four or more, even more preferably five or more, and particularly preferably ten or more polynucleotides) and promoters operably linked thereto (i.e., an expression unit that enables polycistronic mRNA expression). The expression unit can be contained in a genomic region (e.g., a native genomic region that is the natural locus where the polynucleotide encoding the protein is inherently present, or a non-native genomic region that is not the natural locus) or a non-genomic region (e.g., within 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 expression units contained in non-genomic regions include, for example, plasmids, viral vectors, phages, and artificial chromosomes.

[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, constitutive or inducible promoters commonly used in recombinant protein production can be used. Examples of such promoters include the 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 with strong transcriptional activity in the host cell can be used. Examples of promoters with strong transcriptional activity in the 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 that of a wild-type microorganism may be a microorganism (i) comprising a heterologous expression unit containing 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. Therefore, 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 and the promoter constituting the heterologous expression unit are heterologous to the host cell. Therefore, in the present invention, one or both of the polynucleotide encoding the target enzyme and the promoter are derived from an organism other than the host cell (e.g., a prokaryote or eukaryote, or an animal such as a microorganism, insect, plant, or mammal) or a virus, or are artificially synthesized. 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 containing 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 having 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 that of a wild-type microorganism may be (ii) a microorganism containing, in a non-native genomic region or a non-genomic region, an 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 multiple copies, where the multiple copies may be, 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.

[0067] 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 (iv) a microorganism containing a non-natural expression unit in which a mutation has been introduced into a native 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 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, for example, bacteria such as bacteria belonging to the family Enterobacteriaceae, and fungi. Bacteria may also be Gram-positive or Gram-negative. Examples of Gram-positive bacteria include bacteria of the genus Bacillus and Corynebacterium. Preferred examples of Bacillus bacteria include Bacillus subtilis. Preferred examples of Corynebacterium bacteria include Corynebacterium glutamicum. Preferred examples of Gram-negative bacteria include bacteria of the genus Escherichia and Pantoea. Preferred examples of Escherichia bacteria include Escherichia coli. As the Pantoea bacterium, Pantoea ananatis is preferred. As the fungus, microorganisms of the genera Saccharomyces and Schizosaccharomyces are preferred. As the Saccharomyces microorganism, Saccharomyces cerevisiae is preferred. As the Schizosaccharomyces microorganism, Schizosaccharomyces pombe is preferred.

[0070] In the present invention, the host cell used as the transformed microorganism may be, for example, a host with a weakened or defective acylamino acid, fatty acid, or amino acid degradation system. Examples of hosts with a weakened or defective degradation system include hosts with a weakened or defective protein, such as an enzyme, associated with the degradation system, and hosts that produce inhibitors of the enzymes associated with the degradation system. Examples of hosts with a weakened or defective protein, such as an enzyme, associated with the degradation system include hosts containing a mutation in the host genome that reduces or eliminates the expression level of the protein, and hosts containing a mutation in the host genome that reduces or eliminates the activity of the protein. Examples of hosts that produce or enhance inhibitors of the enzymes associated with the degradation system include hosts into which an expression unit for the inhibitor has been introduced by transformation, hosts containing a mutation in the host genome that enhances the expression level of the inhibitor, and hosts containing a mutation in the host genome that enhances the activity of the inhibitor. Examples of proteins, such as enzymes associated with the acylamino acid degradation system include acylase, and examples of proteins, such as enzymes associated with the fatty acid degradation system 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 enzymatic reactions and thereby improve production efficiency. Examples of hosts with enhanced uptake ability include hosts that produce or enhance proteins such as enzymes related to the uptake ability. Examples of hosts that produce or enhance proteins such as enzymes related to the uptake ability include hosts into which an expression unit for the protein has been introduced by transformation, hosts containing mutations in the host genome that enhance the expression level of the protein, and hosts containing mutations in the host genome that enhance 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 above-described transformed microorganism can be prepared by a method using an expression vector (e.g., competent cell method, electroporation method) or a genome modification technique. If 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, if 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, genome editing techniques (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) can be used to integrate the expression unit into the genomic DNA of the host cell and modify the expression unit inherent to the host cell.

[0073] In addition to the above-described minimal expression unit, the expression vector may further contain elements that function in host cells, such as a terminator, a ribosome binding site, and a drug resistance gene. Examples of drug resistance genes include those that are resistant to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0074] The expression vector may further comprise a region that enables 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 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 viral vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. An integrative vector may be a vector that is integrated in its entirety into the genome of a host cell. Alternatively, an integrative vector may be a vector that is integrated only in part (e.g., an expression unit) into the genome of a host cell. 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, the method of the present invention can use an amino group-containing compound or a carboxyl group-containing compound produced in a separate reaction system as a substrate.

[0077] When the method of the present invention is carried out using the enzyme itself (e.g., a purified enzyme), an aqueous solution containing the enzyme can be used as the reaction system. A buffer solution is preferred as the aqueous solution. Examples of buffer solutions include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer. The pH is preferably about 5 to 10. The amounts of the enzyme, amino group-containing compound, and carboxyl group-containing compound (substrate) in the reaction system, as well as 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 a temperature of 20 to 40°C is preferred.

[0078] The method of the present invention may be carried out in combination with an ATP regeneration system. When the method of the present invention is carried out using the above-mentioned enzyme itself (e.g., a purified enzyme), examples of the combination with an ATP regeneration system include a reaction in combination with an ATP regeneration enzyme (e.g., a mixture). Examples of ATP regeneration enzymes 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 above-mentioned enzyme or a processed product thereof, examples of the combination with an ATP regeneration system include using a microorganism with enhanced ATP supply ability as a host. Examples of microorganisms with enhanced ATP supply ability include microorganisms that produce or enhance the above-mentioned ATP regeneration enzyme. Examples of microorganisms that produce or enhance the ATP regeneration enzyme include a host into which an expression unit for the ATP regeneration enzyme has been introduced by transformation, a host containing a mutation in the host genome that enhances the expression level of the ATP regeneration enzyme, and a host containing a mutation in the host genome that enhances the activity of the ATP regeneration enzyme.

[0079] The production of the N-acyl-amino group-containing compound can be confirmed as appropriate, for example, 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 analyzing it with UPLC-MS. [Example]

[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 from Cystobacter fuscus (CfHP, WP_002626336, SEQ ID NO: 9), GH3 auxin-responsive promoter superfamily from Synechococcus sp. PCC 7335 (SsGH3, WP_006458022, SEQ ID NO: 8), indoleacetate-lysine synthetase 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 the resulting plasmid DNA was inserted into the NdeI and XhoI sites within the multiple cloning site of pET-28a(+) (Merck) and purchased from Eurofins Genomics.The plasmids were designated 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 fused with an N-terminal His-tag and thrombin cleavage site.

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

[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). Transformants were inoculated into 100 mL of LB medium containing 25 mg / L kanamycin and cultured at 37°C with shaking in a Sakaguchi flask. When the OD610 reached 0.6, 1 mM IPTG was added, and the culture was continued at 15°C for 24 hours with shaking.

[0084] The plasmid pET-28a-CfHP was introduced into E. coli BL21(DE3), and the transformant was inoculated into 100 mL of LB medium containing 25 mg / L kanamycin and cultured with shaking at 37°C in a Sakaguchi flask. When the OD610 reached 0.2, 1 mM IPTG was added, and the culture was continued with shaking at 15°C 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 at 37°C using a Sakaguchi flask. When the OD610 reached 0.4, 1 mM IPTG was added, and the culture was continued at 15°C for 24 hours with shaking.

[0086] The plasmid pET-28a-SsGH3 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 in a Sakaguchi flask at 37°C. When the OD610 reached 0.2, 1 mM IPTG was added, and the culture was continued with shaking at 15°C for 24 hours.

[0087] 3) Purification of acylamino acid synthetase After the culture was completed, the cells were collected from the resulting culture medium by centrifugation, washed, suspended in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0 or 10 mM imidazole, and then sonicated. 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 5 ml Cartridge (Clontech), equilibrated with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0 or 10 mM imidazole, and adsorbed to the carrier. After washing away unadsorbed proteins with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0 or 10 mM imidazole, the adsorbed proteins were eluted with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 150 mM imidazole at a flow rate of 5 mL / min. 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 culture volume was increased and further purification was performed.

[0088] Example 2: Synthesis of N-caprinoyl amino acid using acyl amino acid synthetase A 0.2 mL reaction mixture containing 50 mM Tris-HCl, 5 mM amino acids, 5 mM sodium caprate, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, and 50 μg / mL purified enzyme, pH 8.0, was incubated for 24 hours at 25°C. After completion of the reaction, 0.8 mL of a 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. A signal corresponding to the molecular weight of N-caprinoyl amino acid was detected.

[0089] The UPLC-MS analysis conditions are as follows. Instrument: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 x 100 mm 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: 2 μL 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 A 0.2 mL reaction mixture 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, and 50 μg / mL purified enzyme, pH 8.0, was incubated at 25°C for 24 hours. After completion of the reaction, 0.8 mL of stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added, and the mixture was filtered and subjected to UPLC-MS analysis. Signals corresponding to the molecular weights of N-caprinoyl amino acid derivatives, N-caprinoyl-D-amino acids, or N-caprinoyl-peptides were detected. The UPLC-MS analysis conditions were as described in Example 2.

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

[0096] [Table 4]

[0097] Example 4: Synthesis of N-lauroylamino acid and N-lauroyl-amino acid derivatives using acylamino acid synthetase A 0.2 mL reaction mixture containing 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, and 200 μg / mL purified enzyme, pH 8.0, was incubated at 25°C for 24 hours. After completion of the reaction, 0.8 mL of a 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. Signals corresponding to the molecular weights of N-lauroyl amino acids and N-lauroyl-amino acid derivatives 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 shown in Table 5 below were combined with amino acids or amino acid derivatives.

[0099] [Table 5]

[0100] Example 5: Synthesis of N-acylamino acids using acylamino acid synthetase 0.1 mL of the reaction mixture, containing 50 mM Tris-HCl, 5 mM amino acids, 5 mM sodium fatty acid salts, 10 mM ATP, 10 mM MgCl2, 1 mM DTT, and 200 μg / mL purified enzyme, pH 8.0, was incubated for 24 hours at 25°C. For AtGH3-6, OsGH3-8, AtGH3-5, and AtGH3-12, L-Asp was used; for CfHP, Gly or L-Ala was used. After the reaction was completed, 0.4 mL of stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added. The mixture was filtered and subjected to UPLC-MS analysis. The N-acyl amino acids produced were quantified by UV detection at 210 nm. The UPLC-MS analysis conditions were as described in Example 2. The analysis results showed that when AtGH3-6 was used, the concentrations of Nα-capryloyl-L-aspartic acid were 3.9 mM, Nα-caprinoyl-L-aspartic acid were 4.5 mM, and Nα-lauroyl-L-aspartic acid were 2.2 mM. When OsGH3-8 was used, the concentrations of Nα-capryloyl-L-aspartic acid were 4.3 mM, Nα-caprinoyl-L-aspartic acid were 4.6 mM, and Nα-lauroyl-L-aspartic acid were 3.5 mM. When AtGH3-5 was used, the concentrations of Nα-capryloyl-L-aspartic acid were 4.1 mM, Nα-caprinoyl-L-aspartic acid were 4.6 mM, and Nα-lauroyl-L-aspartic acid were 2.2 mM. The concentrations of lauroyl-L-aspartic acid were 2.5 mM. When AtGH3-12 was used, the concentrations of Nα-capryloyl-L-aspartic acid, Nα-caprinoyl-L-aspartic acid, and Nα-lauroyl-L-aspartic acid were 1.6 mM, 0.6 mM, and 0.2 mM, respectively. When CfHP was used, the concentrations of Nα-capryloylglycine, Nα-caprinoylglycine, Nα-lauroylglycine, Nα-capryloyl-L-alanine, Nα-capryloyl-L-alanine, Nα-caprinoyl-L-alanine, and Nα-lauroyl-L-alanine were 4.5 mM, 4.6 mM, 0.1 mM, 3.1 mM, 3.6 mM, and 0.4 mM, respectively.

[0101] Example 6: Synthesis of N-acylamino acids using acylamino acid synthetase The reaction mixture consisted of 0.1 mL of 50 mM Tris-HCl, 5 mM amino acids, 5 mM sodium fatty acid (3 mM for sodium palmitate and sodium stearate), 10 mM ATP, 10 mM MgCl2, 1 mM DTT, and 200 μg / mL purified enzyme, pH 8.0, and was shaken for 24 hours at 25°C. When sodium palmitate or sodium stearate was used, the reaction mixture contained 10% (v / v) methanol. 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 subjected to UPLC-MS analysis, detecting 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: 2 μL 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 A 0.25 mL reaction mixture containing 50 mM Tris-HCl, 5 mM amino acids, 5 mM sodium caprate, 10 mM or 0 mM ATP, 10 mM MgCl2, 1 mM DTT, and 50 μg / mL purified enzyme, pH 8.0, was incubated for 24 hours at 25°C. The amino acid used was L-Asp for AtGH3-6 and Gly for CfHP. After the reaction was completed, 0.8 mL of stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added to 0.2 mL of the reaction mixture. The mixture was filtered and then subjected to UPLC-MS analysis. The N-caprinoyl amino acids produced were quantified using UV detection at 210 nm. 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 acids were 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 acids was detected in either AtGH3-6 or CfHP.

[0108] Example 8: Synthesis of N-capryloyl amino acid using acyl amino acid synthetase-expressing bacteria (1) Preparation of various bacterial cell suspensions 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 medium containing 25 mg / L kanamycin and cultured at 37°C using a Sakaguchi flask. When the OD610 reached 0.6, 1 mM IPTG was added, and the culture was continued at 15°C for 24 hours with shaking. BL21(DE3) / pET-28a-CfHP was inoculated into 100 mL of LB containing 25 mg / L kanamycin and cultured with shaking in a Sakaguchi flask at 37°C. When the OD610 reached 0.2, 1 mM IPTG was added, and the culture was continued with shaking at 15°C for 24 hours.

[0109] (Preparation of cell-free extract) After the culture was completed, the cells were collected from 5 mL of the resulting culture medium by centrifugation, washed with 20 mM Tris-HCl (pH 7.6), and then suspended in 1 mL of BugBuster® 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 resulting 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 solution. (Preparation of bacterial cell suspension) After the cultivation was completed, 15 mL of the resulting 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 cultures 0.3 mL of reaction mixture was incubated at 25°C for 24 hours in 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, and 30 μL of bacterial cell solution (cell-free extract, washed bacterial cell solution, or bacterial cell solution), pH 8.0. The amino acid used was L-Asp for AtGH3-6, OsGH3-8, and AtGH3-5, and Gly or L-Ala for CfHP. After the reaction, 0.8 mL of stop solution (1% (v / v) phosphoric acid, 75% (v / v) methanol) was added to 0.2 mL of reaction mixture. The mixture was filtered and subjected to UPLC-MS analysis to detect signals corresponding to N-capryloyl amino acids. 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-acyl amino acids were produced in the absence of ATP (Example 7), but in the reaction using the bacterial cell solution, N-acyl amino acids were produced even in the absence of ATP, suggesting that the enzymatic reaction proceeded using the ATP contained in the bacterial cells.

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

[0114] 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. reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of a GH3 protein to produce an N-acyl-amino group-containing compound; The GH3 protein is: (A) a protein comprising the amino acid sequence of SEQ ID NO: 2; (B) a protein comprising an amino acid sequence of SEQ ID NO: 2, which contains a substitution, deletion, insertion, or addition of 1 or 50 amino acids, and which has 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: 2 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. 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, wherein the amino acid is an L-amino acid or a D-amino acid.

4. The amino acid or peptide is: (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; and (c) gamma-aminobutyric acid; 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 according to any one of claims 1 to 4, wherein the fatty acid is a fatty acid having 6 to 12 carbon atoms.

6. The method according to 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 containing a heterologous expression unit comprising a polynucleotide encoding the GH3 protein and a promoter operably linked thereto; (ii) a microorganism comprising, in a non-native genomic region or a non-genomic region, an 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.

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