Modified enzymes with n-acylation activity

Abstract

To provide enzymes useful for establishing excellent production systems for N-acyl-amino group-containing compounds.SOLUTION: Provided herein is a modified enzyme having N-acylation activity comprising: (A) a modified amino acid sequence composed of amino acid sequence having one or more amino acid mutations in a specific amino acid sequence (1); (B) an amino acid sequence having additional one or several substitution, deletion, insertion or addition of amino acid residues in the amino acid sequence (A); or (C) an amino acid sequence having additional one or more mutated amino acid residues in the amino acid sequence (A) with 90% or more identity thereto, where one of the enzymatic properties of the modified enzyme, such as, (i) N-acylation activity to L-glutamic acid and / or L-aspartic acid and (ii) substrate specificity to L-glutamic acid, is improved as compared with the enzyme of the specific amino acid sequence (1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to modified enzymes having N-acylation activity, etc. [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 enzyme useful for establishing an excellent system for producing N-acyl-amino group-containing compounds, and a method for producing N-acyl-amino group-containing compounds. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have succeeded in creating a modified enzyme with excellent N-acyl-amino group-containing compound production ability by mutating amino acid residues in indole-3-acetic acid-amido synthetase GH3.6 (AtGH3-6) derived from Arabidopsis thaliana, as well as in creating other inventions useful for the production of N-acyl-amino group-containing compounds, thereby completing the present invention.

[0011] That is, the present invention is as follows. [1] The following: (A) a modified amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1, including mutations of one or more amino acid residues selected from the group consisting of N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence containing one or more additional mutations of amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence; Including, A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity toward L-glutamate and / or L-aspartate and substrate specificity toward L-glutamate. [2] The modified enzyme of [1], wherein the mutation comprises one or more amino acid residue mutations selected from the group consisting of N101S, R117P, T122S, I123T, Y134F, Y134V, L137I, V140I, S161P, V174A, Q200E, V231A, V311A, C335S, T336S, M337G, M337A, A339G, S340A, Y344A, Y344G, Y344I, Y344V, R350T, G379D, K388N, L390P, S455T, E483D, Q533R, and C576A. [3] The modified enzyme according to [1] or [2], wherein the N-acylation activity is N-acylation activity against an amino acid. [4] The modified enzyme according to [3], wherein the N-acylation activity for the amino acid has N-acylation activity for L-glutamic acid or L-aspartic acid. [5] A polynucleotide encoding any one of the modified enzymes [1] to [4]. [6] An expression vector comprising the polynucleotide of [5]. [7] A host cell comprising an expression unit of a polynucleotide encoding the modifying enzyme of any one of [1] to [4]. [8] The host cell of [7], wherein the host cell is a microorganism having at least one genetic modification selected from the following: (1) Enhancement of fatty acid supply ability; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) Deficiency or weakening of N-acylamino acid degrading enzyme. [9] The host cell according to [8], wherein the genetic modification for enhancing fatty acid supplying ability is either (a) or (b) below, or both (a) and (b) below: (a) Acyl-CoA synthetase deficiency or weakening; and (b) Acyl-ACP thioesterase enhancement.

[10] The host cell according to any one of [7] to [9], wherein the host cell is a bacterium belonging to the family Enterobacteriaceae.

[11] The host cell of

[10] , wherein the bacterium belonging to the Enterobacteriaceae family is a bacterium of the genus Escherichia or Pantoea.

[12] The host cell of

[11] , wherein the Escherichia bacterium is Escherichia coli and the Pantoea bacterium is Pantoea ananatis.

[13] The following: (A) a modified amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1, including mutations of one or more amino acid residues selected from the group consisting of N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence containing one or more additional mutations of amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence; Including, have N-acylation activity, and A method for producing an N-acyl-amino group-containing compound or a salt thereof, comprising reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of a modified enzyme having an N-acylation activity in which any one of the following properties (i) to (iii) is improved with respect to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1, to produce an N-acyl-amino group-containing compound or a salt thereof: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity toward L-glutamate and / or L-aspartate and substrate specificity toward L-glutamate.

[14] The method according to

[13] , wherein the modified enzyme is a purified enzyme.

[15] The method according to

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

[16] The method according to

[15] , wherein the transformed microorganism is a microorganism that has undergone at least one genetic modification selected from the following: (1) Enhancement of fatty acid supply capacity; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) Deficiency or weakening of N-acylamino acid degrading enzyme.

[17] The method according to

[15] or

[16] , wherein the amino group-containing compound and the carboxyl group-containing compound are produced in the transformed microorganism by culturing the transformed microorganism in the presence of a carbon source.

[18] The following: (1) Enhancement of fatty acid supply capacity; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) N-acylamino acid degrading enzyme deficiency or weakening; and a method for producing an N-acyl-amino group-containing compound or a salt thereof, in which an amino group-containing compound and a carboxyl group-containing compound are bonded by an amide bond, the method comprising culturing, in the presence of a carbon source, a microorganism that has been subjected to at least one genetic modification selected from the group consisting of

[19] The method according to

[18] , wherein the enzyme is a GH3 protein or a PaaK protein.

[20] The GH3 protein is (I) The following: (A) a modified amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1, including mutations of one or more amino acid residues selected from the group consisting of N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence containing one or more additional mutations of amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence; Including, have N-acylation activity, and A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity toward L-glutamate and / or L-aspartate and substrate specificity toward L-glutamate; or (II) The following: (D) a protein comprising the amino acid sequence of SEQ ID NO: 1; (E) a protein comprising an amino acid sequence of SEQ ID NO: 1 containing one or several amino acid substitutions, deletions, insertions, or additions, and having N-acylation activity; or (F) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1 and having N-acylation activity; An enzyme selected from the group consisting of This is the method of

[19] .

[21] Any of the methods

[18] to

[20] , wherein the genetic modification for enhancing fatty acid supplying ability is either (a) or (b) below, or both (a) and (b) below: (a) Acyl-CoA synthetase deficiency or weakening; and (b) Acyl-ACP thioesterase enhancement.

[22] The method according to

[21] , wherein the acyl-ACP thioesterase has thioesterase activity against lauroyl-ACP.

[23] The method of

[21] , wherein the acyl-ACP thioesterase is selected from the following: (i) (i-1) a protein comprising the amino acid sequence of SEQ ID NO: 3, or (i-2) an amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3; (ii) (ii-1) a protein comprising the amino acid sequence of SEQ ID NO: 3, or (ii-2) an amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3, but containing one or several amino acid substitutions, deletions, insertions, or additions, and having acyl-ACP thioesterase activity; and (iii) (iii-1) A protein comprising the amino acid sequence of SEQ ID NO: 3, or (iii-2) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having acyl-ACP thioesterase activity.

[24] The method according to any one of

[18] to

[23] , wherein the carbon source is a sugar.

[25] The method according to

[24] , wherein the sugar is glucose.

[26] The method according to any one of

[18] to

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

[27] The method according to

[26] , wherein the bacterium belonging to the Enterobacteriaceae family is a bacterium of the genus Escherichia or Pantoea.

[28] The method according to

[27] , wherein the Escherichia bacterium is Escherichia coli and the Pantoea bacterium is Pantoea ananatis.

[29] The method according to any one of

[13] to

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

[30] The method according to

[29] , wherein the fatty acid is a fatty acid having 8 to 18 carbon atoms.

[31] The method according to

[29] , wherein the fatty acid is a fatty acid having 12 carbon atoms.

[32] Any of the methods

[29] to

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

[33] The method according to any one of

[13] to

[32] , wherein the amino group-containing compound is an amino acid, and the enzyme has an N-acylation activity for the amino acid.

[34] The method according to

[33] , wherein the amino acid is L-glutamic acid or L-aspartic acid.

[35] The method according to any one of

[13] to

[34] , wherein the carboxyl group-containing compound is lauric acid, and the N-acyl-amino group-containing compound is Nα-lauroyl-L-glutamic acid or Nα-lauroyl-L-aspartic acid.

[36] The following: (1) Enhancement of fatty acid supply ability; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) N-acylamino acid degrading enzyme deficiency or weakening; and further comprising an expression unit for a polynucleotide encoding an enzyme capable of forming an amide bond by bonding a carboxyl group and an amino group in an ATP-dependent manner, and capable of producing an N-acyl-amino group-containing compound or a salt thereof when cultured in the presence of a carbon source.

[37] A surfactant containing an N-acyl-amino group-containing compound or a salt thereof produced by the method according to any one of

[13] to

[35] .

[38] The surfactant according to

[37] , wherein the N-acyl-amino group-containing compound or its salt comprises an N-monounsaturated acyl-amino group-containing compound having a monounsaturated acyl having 10 to 16 carbon atoms or its salt.

[39] A surfactant containing an N-monounsaturated acyl-amino group-containing compound having a monounsaturated acyl having 10 to 16 carbon atoms, or a salt thereof.

[40] The surfactant according to any one of

[37] to

[39] , wherein the N-monounsaturated acyl-amino group-containing compound is a compound represented by the following general formula (1): [ka] (In the general formula (1), l is an integer of 1 or 2, and m is an integer of 0 to 6.)

[41] The surfactant according to any one of

[37] to

[40] , wherein the stereochemistry of the N-monounsaturated acyl-amino group-containing compound is cis.

[42] The surfactant according to any one of

[37] to

[41] , wherein the N-acyl-amino group-containing compound or its salt is N-monounsaturated acylglutamic acid or its salt.

[43] Component (A) N-monounsaturated acyl acidic amino acid (wherein the unsaturated acyl is an acyl having 10 to 16 carbon atoms) or a salt thereof, and A composition comprising component (B) an N-saturated acyl acidic amino acid or a salt thereof. [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. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the structure of the plasmid pMW118-Sce-Km. [Figure 2] FIG. 2 is a diagram showing the structure of the plasmid pMW118-Ptac-UcTEopt. [Figure 3] FIG. 3 shows the structure of the plasmid pMW118-PlacUV5-lacI-UcTEopt. [Figure 4]FIG. 4 shows (1) the amino acid sequence of indole-3-acetic acid-amido synthetase GH3.6 (AtGH3-6) derived from Arabidopsis thaliana (SEQ ID NO: 1), and (2) the amino acid sequence of medium-chain acyl-ACP thioesterase (UcTE) derived from California bay (Umbellularia californica) (SEQ ID NO: 3). DETAILED DESCRIPTION OF THE INVENTION

[0014] (modified enzyme) The present invention provides modified enzymes that have N-acylation activity.

[0015] The modified enzymes of the present invention are enzymes modified based on Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.6 (Q9LSQ4, hereinafter referred to as "AtGH3-6"). AtGH3-6 was discovered as an enzyme that forms an amide bond by combining the carboxyl group of indole-3-acetic acid with the amino group of a specific amino acid in an ATP-dependent manner (Plant Cell 17:616-627 (2005)). The present inventors also found that it has N-acylation activity, including the ability to form an amide bond between a fatty acid and an amino acid. The modified enzymes of the present invention are enzymes with N-acylation activity that have been modified from AtGH3-6 to improve their properties related to the ability to produce N-acyl-amino group-containing compounds.

[0016] The modified enzyme of the present invention is the following modified enzyme: (A) a modified amino acid sequence consisting of an amino acid sequence of SEQ ID NO: 1 (wild-type AtGH3-6) containing one or more mutations selected from a group of predetermined amino acid residue mutations; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence containing one or more additional mutations of amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence; Including, A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity toward L-glutamate and / or L-aspartate and substrate specificity toward L-glutamate.

[0017] Alternatively expressed, the modified enzyme of the present invention is the following modified enzyme: the below described: (A') the amino acid sequence shown in SEQ ID NO: 1; (B') an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence shown in SEQ ID NO: 1; or (C') an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence contains one or more amino acid residue mutations selected from a group of predetermined amino acid residue mutations, A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity toward L-glutamate and / or L-aspartate and substrate specificity toward L-glutamate.

[0018] In the amino acid sequences (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 the number of residues that does not significantly impair the activity (e.g., N-acylation activity) of the modified enzyme. The number referred to by the term "one or several" is, for example, 1 to 60 or 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).

[0019] The percent identity between amino acid sequences (C) and (C') is 90% or greater. Preferably, the identity may be 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. The percent identity of a polypeptide (protein) can be calculated using the blastp algorithm. More specifically, the percent identity of a polypeptide can be calculated 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). The percent identity of a polynucleotide (gene) can be calculated 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).

[0020] "N-acylation activity" (sometimes referred to as "N-acylase activity") refers to the activity of linking an amino group-containing compound and a carboxyl group-containing compound as substrates via an amide bond to produce an N-acyl-amino group-containing compound. N-acylation activity may be, for example, the activity of producing an N-acylamino acid from an amino acid (e.g., an α-L-amino acid, as described below) and a fatty acid (e.g., a saturated fatty acid, as described below). Preferably, the activity of producing Nα-lauroyl-L-glutamic acid or Nα-lauroyl-L-aspartic acid from L-glutamic acid or L-aspartic acid and lauric acid may be used as an indicator. "Having N-acylation activity" may mean, when the activity is measured under specific measurement conditions, that the activity is, 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 equivalent (i.e., 100%) or more of the activity of an enzyme consisting of the amino acid sequence of SEQ ID NO: 1. The following specific measurement conditions can be used: The enzyme to be measured is prepared as a purified enzyme, and 0.2 mL of a reaction solution containing 50 mM Tris-HCl, 5 mM amino acid (e.g., L-glutamic acid or L-aspartic acid), 5 mM sodium fatty acid (e.g., sodium laurate), 10 mM ATP, 10 mM MgCl2, 1 mM DTT, and 50 μg / mL purified enzyme, pH 8.0, is incubated at 25°C for 24 hours. After the reaction is complete, 0.8 mL of a reaction stop solution (1.4% (w / v) phosphoric acid, 75% (v / v) methanol) is added, and the supernatant after centrifugation is subjected to UPLC-MS analysis. The N-acylation activity is evaluated by measuring the signal corresponding to the molecular weight of N-acylamino acid (e.g., Nα-lauroyl-L-glutamic acid or Nα-lauroyl-L-aspartic acid).

[0021] Amino acid sequences (B), (B'), (C), and (C') may contain mutations at sites within the catalytic domain and at sites outside the catalytic domain, as long as the modified enzyme retains the desired properties. The positions of amino acid residues that may be mutated so that the modified enzyme retains 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 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, 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.

[0022] 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.

[0023] The modified enzymes of the present invention may also be fusion proteins 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 for purifying the target protein such as glutathione-S-transferase, maltose-binding protein, and mutant forms 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 recognized by proteases that cleave the purification tag (e.g., thrombin recognition sequence, TEV protease recognition sequence), peptide moieties with other functions (e.g., full-length proteins or portions thereof), and linkers.

[0024] "Predetermined amino acid residue mutations" in the amino acid sequence set forth in SEQ ID NO: 1 or any of the amino acid sequences (A'), (B'), and (C') are mutations that improve the N-acyl-amino group-containing compound-producing ability of the modified enzyme. Examples of "predetermined amino acid residue mutations" include mutations in the following amino acid residues: N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specifically, "mutations of predetermined amino acid residues" include substitutions of the following amino acid residues: N101S, R117P, T122S, I123T, Y134F, Y134V, L137I, V140I, S161P, V174A, Q200E, V231A, V311A, C335S, T336S, M337G, M337A, A339G, S340A, Y344A, Y344G, Y344I, Y344V, R350T, G379D, K388N, L390P, S455T, E483D, Q533R, and C576A. The number of "specific amino acid residue mutations" contained in the modified enzyme of the present invention may be one or more, for example, 1 to 26, preferably 1 to 20, more preferably 1 to 15, and even more preferably 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0025] The properties related to the ability to produce an N-acyl-amino group-containing compound that are improved by "mutating a specific amino acid residue" include N-acylation activity for a specific amino acid substrate and substrate specificity for a specific amino acid substrate. The degree of improvement is shown by comparison with an enzyme consisting of the amino acid sequence of SEQ ID NO: 1, and may also be measured by comparison between fusion proteins linked to a heterologous moiety via a peptide bond.

[0026] Examples of N-acylation activity toward specific amino acid substrates include N-acylation activity toward L-glutamic acid and N-acylation activity toward L-aspartic acid. The carboxyl group-containing compound used as a substrate for measuring N-acylation activity may be, for example, a fatty acid, preferably a saturated fatty acid, and more preferably lauric acid. Measurement of N-acylation activity can be performed, for example, under the measurement conditions described above. The degree of improvement in N-acylation activity toward specific amino acid substrates is not particularly limited as long as it exceeds the N-acylation activity of the enzyme consisting of the amino acid sequence of SEQ ID NO: 1, but is, for example, 1.1-fold or more, preferably 1.2-fold or more, more preferably 1.3-fold or more, even more preferably 1.5-fold or more, and most preferably 2-fold or more.

[0027] Mutations that improve N-acylation activity toward L-glutamic acid or L-aspartic acid include mutations of the following amino acid residues: N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specifically, mutations that improve N-acylation activity toward L-glutamic acid or L-aspartic acid include substitutions of the following amino acid residues: N101S, R117P, T122S, I123T, Y134F, Y134V, L137I, V140I, S161P, V174A, Q200E, V231A, V311A, C335S, T336S, M337G, M337A, A339G, S340A, Y344A, Y344G, Y344I, Y344V, R350T, G379D, K388N, L390P, S455T, E483D, Q533R, and C576A.

[0028] Substrate specificity for a specific amino acid substrate includes substrate specificity for L-glutamic acid. Substrate specificity for L-glutamic acid can be expressed as the ratio of N-acylation activity for L-glutamic acid to that for another amino acid (e.g., L-aspartic acid) when the same carboxyl group-containing compound substrate is used. The same carboxyl group-containing compound substrate may be, for example, a fatty acid, preferably a saturated fatty acid, and more preferably lauric acid. N-acylation activity can be measured, for example, under the measurement conditions described above. The degree of improvement in substrate specificity for a specific amino acid substrate is not particularly limited as long as it exceeds the substrate specificity of an enzyme consisting of the amino acid sequence of SEQ ID NO: 1, but is, for example, 1.1-fold or more, preferably 1.2-fold or more, more preferably 1.3-fold or more, even more preferably 1.5-fold or more, and most preferably 2-fold or more.

[0029] Mutations that improve substrate specificity for L-glutamate include mutations of the following amino acid residues: N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specifically, mutations that improve substrate specificity for L-glutamic acid include substitutions of the following amino acid residues: N101S, R117P, T122S, I123T, Y134F, Y134V, L137I, V140I, S161P, V174A, Q200E, V231A, V311A, C335S, T336S, M337G, M337A, A339G, S340A, Y344A, Y344G, Y344I, Y344V, R350T, G379D, K388N, L390P, S455T, E483D, Q533R, and C576A.

[0030] (Polynucleotide) The present invention also provides a polynucleotide encoding the modified enzyme of the present invention. The polynucleotide of the present invention may be DNA or RNA, but is preferably DNA.

[0031] Preferably, the polynucleotide encoding the enzyme used in the present invention may be a polynucleotide encoding: (a) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 (wild-type AtGH3-6); (b) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2; (c) a polynucleotide containing a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 2; and (d) a degenerate variant of a polynucleotide selected from the group consisting of (a) to (c). In a polynucleotide selected from the group consisting of: It includes mutations in the base sequence corresponding to mutations in amino acid residues corresponding to mutations in one or more amino acid residues selected from the group of predetermined amino acid residue mutations described above, A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamate and / or L-aspartate; (ii) substrate specificity for L-glutamate; or (iii) N-acylation activity for L-glutamic acid and / or L-aspartic acid, and substrate specificity for L-glutamic acid. The nucleotide sequence of SEQ ID NO: 2 encodes the amino acid sequence of SEQ ID NO: 1.

[0032] 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.

[0033] In the above polynucleotide (c), the percent identity of the base sequence to the base sequence of SEQ ID NO: 2 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.

[0034] 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.

[0035] 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.

[0036] [Table 1]

[0037] 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.

[0038] 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.

[0039] (expression vector) The present invention provides an expression vector, which comprises a polynucleotide of the present invention or a polynucleotide encoding a modified enzyme of the present invention.

[0040] 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.

[0041] 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.

[0042] 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, phosphinothricin, and chloramphenicol.

[0043] 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.

[0044] 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.

[0045] (host cell) The present invention provides a host cell. The host cell of the present invention comprises an expression unit of a polynucleotide encoding the modified enzyme of the present invention. The host cell of the present invention preferably comprises an expression unit comprising a polynucleotide encoding the modified enzyme of the present invention and a promoter operably linked thereto. The host cell of the present invention is preferably a microorganism.

[0046] The host cell of the present invention is preferably a transformed microorganism. Examples of the host cell of the present invention include bacteria, such as bacteria belonging to the Enterobacteriaceae family, and fungi. The bacteria may also be Gram-positive or Gram-negative. Examples of Gram-positive bacteria include bacteria of the genus Bacillus and Corynebacterium. Examples of Bacillus bacteria include Bacillus subtilis. Examples of Corynebacterium bacteria include Corynebacterium glutamicum. Examples of Gram-negative bacteria include bacteria of the genus Escherichia and Pantoea. 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. The host cell of the present invention is preferably a bacterium belonging to the family Enterobacteriaceae, more preferably a bacterium of the genus Escherichia or a bacterium of the genus Pantoea, and even more preferably Escherichia coli and Pantoea ananatis.

[0047] In one embodiment, the host cells of the present invention can be used to produce an N-acyl-amino group-containing compound or its salt using the modified enzyme of the present invention produced in the host cells, using the host cells themselves (e.g., host cell culture) or processed products thereof (e.g., host cell lysates, lysates, lyophilized products). In another embodiment, the host cells of the present invention can be used to obtain the modified enzyme of the present invention as an unpurified, crude, or purified enzyme.

[0048] When the host cell of the present invention is used for producing an N-acyl-amino group-containing compound or a salt thereof, the host cell of the present invention may be a host with enhanced ability to uptake amino acids and / or fatty acids, for example, 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 associated with the uptake ability. Examples of hosts that produce or enhance proteins such as enzymes associated with 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. Such host cells can be used to produce an N-acyl-amino group-containing compound (e.g., an N-acylamino acid) or a salt thereof by culturing the host cell in a culture medium containing an amino group-containing compound (e.g., an amino acid) and / or a carboxyl group-containing compound (e.g., a fatty acid). Furthermore, such host cells can be used for producing an N-acyl-amino group-containing compound (e.g., an N-acylamino acid) or a salt thereof (direct fermentation method) by culturing the host cells in a culture medium containing a carbon source (e.g., a sugar such as glucose).

[0049] The host cell of the present invention may be a host cell that has been genetically modified in at least one way selected from the following, for example, to suppress loss of substrates for enzymatic reactions and / or to promote the supply of substrates for enzymatic reactions or to suppress loss of products: (1) Enhancement of fatty acid supply ability; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) Deficiency or weakening of N-acylamino acid degrading enzyme.

[0050] A host cell that has been genetically modified to be effective in enhancing fatty acid supplying ability may be a host cell in which the fatty acid degradation system is weakened or deleted and / or the fatty acid synthesis system is enhanced.

[0051] Examples of host cells in which the fatty acid degradation system is weakened or deficient include host cells in which proteins such as enzymes associated with the degradation system are weakened or deficient, and host cells that produce inhibitors of proteins such as enzymes associated with the degradation system. Examples of host cells in which proteins such as enzymes related to the fatty acid degradation system are weakened or deleted include host cells containing a mutation in the host cell genome that reduces or eliminates the expression level of the protein, and host cells containing a mutation in the host cell genome that reduces or eliminates the activity of the protein. Examples of host cells that produce or enhance inhibitors of proteins such as enzymes involved in the fatty acid degradation system include host cells into which an expression unit for the inhibitor has been introduced by transformation, host cells containing a mutation in the host cell genome that enhances the expression level of the inhibitor, and host cells containing a mutation in the host cell genome that enhances the activity of the inhibitor. More specifically, examples of proteins such as enzymes involved in the fatty acid degradation system include acyl-CoA synthetase (fadD), acyl-CoA dehydrogenase (fadE), enoyl-CoA hydratase (fadB, fadJ), 3-hydroxyacyl-CoA dehydrogenase (fadB, fadJ), and 3-ketoacyl-CoA thiolase (fadA, fadI). The gene name is shown in parentheses after the enzyme name (the same applies to the following descriptions). Among these enzymes, acyl-CoA synthetase is preferred.

[0052] Examples of host cells with an enhanced fatty acid synthesis system include host cells that produce or enhance proteins such as enzymes associated with the synthesis system. Examples of host cells that produce or enhance proteins such as enzymes related to the above-mentioned synthesis system include host cells into which an expression unit for the above-mentioned protein has been introduced by transformation, host cells that contain a mutation in the host cell genome that enhances the expression level of the above-mentioned protein, and host cells that contain a mutation in the host cell genome that enhances the activity of the above-mentioned protein. More specifically, examples of proteins such as enzymes involved in the fatty acid synthesis system include acyl-ACP thioesterase, acetyl-CoA carboxylase (accABCD), malonyl-CoA-ACP transacylase (fabD), β-ketoacyl-ACP synthase III (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydratase (fabZ), enoyl-ACP reductase (fabI), β-ketoacyl-ACP synthase I (fabB), and β-ketoacyl-ACP synthase II (fabF). Among these enzymes, for example, acyl-ACP thioesterase is preferred.

[0053] Preferably, the genetic modification for enhancing the fatty acid supplying ability may be either (a) or (b) below, or both (a) and (b) below. (a) Acyl-CoA synthetase deficiency or weakening; and (b) Acyl-ACP thioesterase enhancement.

[0054] Examples of acyl-CoA synthetase deficiency or attenuation include deletion or introduction of a mutation into the fadD gene in the host cell genome.

[0055] Examples of enhancing acyl-ACP thioesterase include introducing an acyl-ACP thioesterase expression unit into the host cell.

[0056] The acyl-ACP thioesterase may be, for example, an acyl-ACP thioesterase having thioesterase activity toward lauroyl-ACP. Alternatively, the acyl-ACP thioesterase may be, for example, a protein selected from the following: (i) (i-1) a protein comprising the amino acid sequence of SEQ ID NO: 3, or (i-2) an amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3; (ii) (ii-1) a protein comprising the amino acid sequence of SEQ ID NO: 3, or (ii-2) an amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3, but containing one or several amino acid substitutions, deletions, insertions, or additions, and having acyl-ACP thioesterase activity; and (iii) (iii-1) A protein comprising the amino acid sequence of SEQ ID NO: 3, or (iii-2) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having acyl-ACP thioesterase activity.

[0057] A host cell that has been genetically modified to be effective in enhancing amino acid supplying ability may be a host cell with a weakened or defective amino acid degradation system and / or a host cell with an enhanced amino acid synthesis system.

[0058] Examples of host cells in which the amino acid degradation system is weakened or deficient include host cells in which proteins such as enzymes associated with the amino acid degradation system are weakened or deficient, and host cells that produce inhibitors of proteins such as enzymes associated with the amino acid degradation system. Examples of host cells in which proteins such as enzymes involved in the amino acid degradation system are weakened or deleted include host cells containing a mutation in the host cell genome that reduces or eliminates the expression level of the protein, and host cells containing a mutation in the host cell genome that reduces or eliminates the activity of the protein. Examples of host cells that produce or enhance inhibitors of proteins such as enzymes involved in the amino acid degradation system include host cells into which an expression unit for the inhibitor has been introduced by transformation, host cells containing a mutation in the host cell genome that enhances the expression level of the inhibitor, and host cells containing a mutation in the host cell genome that enhances the activity of the inhibitor. More specifically, proteins such as enzymes involved in the amino acid degradation system may be enzymes that catalyze a reaction that branches off from the biosynthetic pathway of a target amino acid (e.g., L-glutamic acid) to produce a compound other than the target amino acid (e.g., L-glutamic acid). Examples of such enzymes include α-ketoglutarate dehydrogenase (sucA), isocitrate lyase (aceA), succinate dehydrogenase (sdhABCD), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyacid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and 1-pyrroline-5-carboxylate dehydrogenase (putA). Among these enzymes, α-ketoglutarate dehydrogenase is preferred.

[0059] Examples of host cells with an enhanced amino acid synthesis system include host cells that produce or enhance proteins such as enzymes involved in the amino acid synthesis system. Examples of host cells that produce or enhance proteins such as enzymes related to the amino acid synthesis system include host cells into which an expression unit for the protein of the amino acid has been introduced by transformation, host cells containing a mutation in the host cell genome that enhances the expression level of the protein, and host cells containing a mutation in the host cell genome that enhances the activity of the protein. More specifically, proteins such as enzymes related to the synthesis of amino acids (e.g., glutamic acid) include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate kinase (pykA, pykF), pyruvate dehydrogenase (aceEF, lpdA), ), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase.

[0060] A host cell that has been genetically modified to be effective in enhancing ATP supply capacity may be a host cell with a weakened or defective ATP degradation system and / or a host cell with an enhanced ATP synthesis system.

[0061] Examples of host cells in which the ATP degradation system is weakened or deficient include host cells in which proteins such as enzymes associated with the ATP degradation system are weakened or deficient. Examples of host cells in which proteins such as enzymes related to the ATP degradation system are weakened or deleted include host cells containing a mutation in the host cell genome that reduces or eliminates the expression level of the protein, and host cells containing a mutation in the host cell genome that reduces or eliminates the activity of the protein.

[0062] Examples of host cells with an enhanced ATP synthesis system include host cells that produce or enhance proteins such as enzymes associated with the synthesis system. Examples of host cells that produce or enhance proteins such as enzymes related to the ATP synthesis system include host cells into which an expression unit for the protein has been introduced by transformation, host cells that contain a mutation in the host cell genome that enhances the expression level of the protein, and host cells that contain a mutation in the host cell genome that enhances the activity of the protein.

[0063] Examples of effective genetic modifications for the deficiency or attenuation of N-acylamino acid degrading enzymes include deficiency or attenuation of acylase, such as deletion or mutation of the acylase gene in the host cell genome.

[0064] Such host cells can be used as transformed microorganisms (e.g., microbial cultures) that produce the enzymes used in the present invention, or processed products thereof (e.g., microbial lysates, lysates, lyophilized products) to produce an N-acyl-amino group-containing compound (e.g., N-acylamino acid) or its salt (enzymatic method) in a reaction solution containing an amino group-containing compound (e.g., amino acid) and a carboxyl group-containing compound (e.g., fatty acid). Such host cells can be cultured in a culture solution containing an amino group-containing compound (e.g., amino acid) and / or a carboxyl group-containing compound (e.g., fatty acid) to produce an N-acyl-amino group-containing compound (e.g., N-acylamino acid) or its salt (enzymatic method). Furthermore, such host cells have an enhanced metabolic pathway for producing an amino group-containing compound (e.g., amino acid) and / or a carboxyl group-containing compound (e.g., fatty acid) from a carbon source (e.g., sugars such as glucose), and therefore can be used to produce an N-acyl-amino group-containing compound (e.g., N-acylamino acid) or its salt by culturing the host cells in a culture solution containing a carbon source (direct fermentation method).

[0065] 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.

[0066] (Method for producing an N-acylamino group-containing compound or its salt) The present invention provides a method for producing an N-acyl-amino group-containing compound or a salt thereof. The method of the present invention comprises reacting an amino group-containing compound with a carboxyl group-containing compound in the presence of a modified enzyme having N-acylation activity to produce the N-acyl-amino group-containing compound or a salt thereof.

[0067] The modified enzyme used in the method of the present invention may be the modified enzyme of the present invention described above.

[0068] (Amino group-containing compounds) 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.

[0069] 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.

[0070] Examples of amino group-containing compounds having a carboxyl group as an anionic group include amino acids and peptides.

[0071] 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.

[0072] 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.

[0073] Examples of amino group-containing compounds having a sulfonic acid group as an anionic group include taurine, N-methyltaurine, and cysteic acid.

[0074] Examples of amino group-containing compounds having a sulfate group as an anionic group include O-sulfoserine and O-sulfothreonine.

[0075] Examples of amino group-containing compounds having a phosphate group as an anionic group include ethanolamine phosphate, phosphoserine, and phosphothreonine.

[0076] The amino group-containing compound that can be used in the method of the present invention is preferably an acidic amino acid. "Acidic amino acid" refers to an amino acid having an acidic side chain. Examples of acidic amino acids include glutamic acid and aspartic acid, with glutamic acid being preferred.

[0077] (Carboxyl group-containing compound) The carboxyl group-containing compound that can be used in the method of the present invention is a compound containing 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, indole carboxylic acids, and mixtures thereof. Fatty acids are preferred as the carboxyl group-containing compound.

[0078] The fatty acid may be, for example, a fatty acid having 8 to 18 carbon atoms, preferably a fatty acid having 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), decenoic acid (C10:1), undecylic acid (C11), lauric acid (C12), dodecenoic acid (C12:1), tridecylic acid (C13), myristic acid (C14), tetradecenoic acid (C14:1) (e.g., myristoleic acid), pentadecylic acid (C15), palmitic acid (C16), Examples include hexadecenoic acid (C16:1) (e.g., palmitoleic acid, sapienic acid), margaric acid (C17), stearic acid (C18), octadecenoic acid (C18:1) (e.g., oleic acid, vaccenic acid), linoleic acid (C18:2), and linolenic acid (C18:3) (e.g., α-linolenic acid, γ-linolenic acid) (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.

[0079] The fatty acid may be a saturated fatty acid. Among the above-mentioned fatty acids, examples of the saturated fatty acid 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. As the saturated fatty acid, lauric acid is preferred. The fatty acid may be an unsaturated saturated fatty acid. Among the above fatty acids, unsaturated fatty acids include decenoic acid (C10:1), dodecenoic acid (C12:1), tetradecenoic acid (C14:1) (e.g., myristoleic acid), hexadecenoic acid (C16:1) (e.g., palmitoleic acid, sapienic acid), octadecenoic acid (C18:1) (e.g., oleic acid, vaccenic acid), linoleic acid (C18:2), and linolenic acid (C18:3) (e.g., α-linolenic acid, γ-linolenic acid) (the number in parentheses indicates the number of carbon atoms). As the unsaturated fatty acid, dodecenoic acid (C12:1) or hexadecenoic acid (C16:1) is preferred. The fatty acid may also be a mixture thereof.

[0080] Aromatic carboxylic acids include benzoic acid, phthalic acid, isophthalic acid, terephthalic acid, salicylic acid, gallic acid, cinnamic acid, and mixtures thereof.

[0081] (N-acyl-amino group-containing compound or its salt) The N-acyl-amino group-containing compound or its salt 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 or its salt is produced by reacting the amino group-containing compound with the carboxyl group-containing compound in the presence of the enzyme. The amino group reacting 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. The N-acyl-amino group-containing compound or its salt produced by the method of the present invention is preferably Nα-acyl-L-glutamic acid or Nα-acyl-L-aspartic acid or its salt, and more preferably Nα-lauroyl-L-glutamic acid or Nα-lauroyl-L-aspartic acid or its salt. Salts of N-acyl-amino group-containing compounds include, for example, inorganic salts and organic salts. Examples of inorganic salts include salts of metals (e.g., monovalent metals such as lithium, sodium, potassium, rubidium, and cesium, and divalent metals such as calcium, magnesium, and zinc) and salts of inorganic bases (e.g., ammonia). Examples of organic salts include salts of organic bases (e.g., ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, triethanolamine, lysine, arginine, histidine, and ornithine).

[0082] As used herein, "acyl having n carbon atoms" refers to n-1 H m It refers to acyl represented by -CO- (hydrogen atoms may be substituted), where m is determined appropriately depending on the number of carbon atoms and the presence or absence of unsaturated bonds.

[0083] In the present specification, the term "N-monounsaturated acyl-amino group-containing compound (wherein the unsaturated acyl is a monounsaturated acyl having 10 to 16 carbon atoms)" refers to a C n-1 H m -CO-NH-CHR-COOH (H m may be substituted. m is as defined above. R represents the side chain of an acidic amino acid. ) That is, an "N-monounsaturated acyl-amino group-containing compound" refers to a compound in which one hydrogen atom on the amino group of an acidic amino acid is substituted with an unsaturated acyl group. The "N-monounsaturated acyl-amino group-containing compound" may be a free form of an N-monounsaturated acyl-amino group-containing compound, or a salt of an N-monounsaturated acyl-amino group-containing compound.

[0084] As used herein, N-monounsaturated acyl-amino group-containing compounds are conveniently referred to as C n-1 H m In this specification, "unsaturated acyl having n carbon atoms" and the fatty acids derived therefrom are sometimes expressed as "Cn:m." For example, "N-dodecenonyl acidic amino acid (C12:1)" refers to a compound (C12:1) in which one hydrogen atom on the amino group of an acidic amino acid is substituted with a dodecenonyl group, which is an acyl group derived from dodecenoic acid (C12:1). 11 H 21 CO—NH—CHR—COOH, where R represents the side chain of an acidic amino acid.

[0085] When the N-acyl-amino group-containing compound or a salt thereof produced by the method of the present invention contains an N-monounsaturated acyl-amino group-containing compound or a salt thereof, examples of the N-monounsaturated acyl-amino group-containing compound include N-decenoyl acidic amino acid (C10:1), N-dodecenoyl acidic amino acid (C12:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1), with N-dodecenoyl acidic amino acid (C12:1) or N-hexadecenoyl acidic amino acid (C16:1) being preferred, N-dodecenoyl glutamic acid (C12:1) or N-hexadecenoyl glutamic acid (C16:1) being more preferred, and N-dodecenoyl glutamic acid (C12:1) being even more preferred.

[0086] The N-monounsaturated acyl-amino group-containing compound may be one type of N-monounsaturated acyl-amino group-containing compound, or may contain two or more types of N-monounsaturated acyl-amino group-containing compounds. That is, the N-monounsaturated acyl-amino group-containing compound may contain one or more amino acids selected from the group consisting of N-decenoyl acidic amino acid (C10:1), N-dodecenoyl acidic amino acid (C12:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1). When two or more types of N-monounsaturated acyl-amino group-containing compounds are used, a combination of N-dodecenoyl acidic amino acid (C12:1) with one or more selected from the group consisting of N-decenoyl acidic amino acid (C10:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1) is preferred, and from the viewpoint of improving foam quality, a combination of N-dodecenoyl acidic amino acid (C12:1) with N-hexadecenoyl acidic amino acid (C16:1) is more preferred.

[0087] The N-monounsaturated acyl-amino group-containing compound is preferably a compound represented by the following general formula (1).

[0088] [ka] (In general formula (1), l is an integer of 1 or 2, and m is an integer of 0 to 6.)

[0089] The stereochemistry (unsaturated double bond site) of the N-monounsaturated acyl-amino group-containing compound may be either cis or trans, although cis is preferred from the viewpoint of suppressing an increase in viscosity and achieving a low viscosity that is easy to handle.

[0090] (Enzymatic production of N-acyl-amino group-containing compounds) Recombinant proteins can be used as the enzymes used in the methods of the present invention. 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, roughly purified, or purified enzymes. These enzymes may also be used as immobilized proteins immobilized on a solid phase in the reaction. The enzymes used in the present invention can be used in the form of transformed microorganisms that produce the enzymes (e.g., microbial cultures) or processed products thereof (e.g., disrupted, lysed, or lyophilized microorganisms).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] (Production of N-acyl-amino group-containing compounds by microbial culture method) In the method of the present invention, the reaction in the presence of the enzyme may be carried out using a transformed microorganism (eg, a microbial culture) that produces the enzyme.

[0100] When the method of the present invention is carried out using a culture of the above-mentioned transformed microorganism, the transformed microorganism may be a host with an enhanced ability to take up, for example, amino acids and / or fatty acids, in order to improve the efficiency of supplying substrates for the enzymatic reaction and thereby improve production efficiency. Examples of hosts with enhanced uptake ability include those described above. In this case, by culturing the transformed microorganism in a culture medium containing an amino group-containing compound (e.g., an amino acid) and / or a carboxyl group-containing compound (e.g., a fatty acid), both substrates are taken up by the transformed microorganism, and an amide bond is formed by the enzyme produced in the transformed microorganism, thereby producing the desired N-acyl-amino group-containing compound (e.g., an N-acyl amino acid).

[0101] Furthermore, in order to suppress loss of substrates for enzymatic reactions and / or to promote the supply of substrates for enzymatic reactions or to suppress loss of products, the transformed microorganism may be subjected to at least one or more genetic modifications selected from the following: (1) Enhancement of fatty acid supply capacity; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) Deficiency or weakening of N-acylamino acid degrading enzyme. Such a transformed microorganism may preferably have undergone genetic modification that is effective in enhancing fatty acid supplying ability, and may be a transformed microorganism in which the fatty acid degradation system is weakened or deleted and / or the fatty acid synthesis system is enhanced. Examples of the transformed microorganism (host) in which the degradation system is weakened or deleted and / or the synthesis system is enhanced include those described above. Such a transformed microorganism preferably includes a microorganism in which at least one or more genetic modifications selected from the following are performed: (a) Acyl-CoA synthetase deficiency or weakening; and (b) Acyl-ACP thioesterase enhancement. The details of the genetic modifications (a) and (b) are as described above.

[0102] When an N-acyl-amino group-containing compound is produced by culturing a transformed microorganism, the culture conditions for the transformed microorganism are not particularly limited. For example, the culture conditions described above can be used in which the transformed microorganism is cultured in a medium further containing a predetermined amount of an amino group-containing compound and / or a carboxyl group-containing compound.

[0103] (Production of N-acyl-amino group-containing compounds by direct fermentation) The present invention provides a method for producing an N-acyl-amino group-containing compound by direct fermentation. The term "direct fermentation" refers to a method for producing an N-acyl-amino group-containing compound (e.g., an N-acylamino acid) by culturing a transformed microorganism in a culture medium containing a carbon source as a raw material and producing the desired N-acyl-amino group-containing compound (e.g., an N-acylamino acid) from the carbon source. This method is based on the principle that a carbon source is taken up by the microorganism, metabolized within the microorganism into a carboxyl group-containing compound (e.g., a fatty acid) and an amino group-containing compound (e.g., an amino acid), and the carboxyl group-containing compound (e.g., a fatty acid) and the amino group-containing compound (e.g., an amino acid) produced by metabolism are linked by an amide bond by the action of an enzyme with N-acylation activity in the microorganism to produce the desired N-acyl-amino group-containing compound (e.g., an N-acylamino acid).

[0104] For example, the direct fermentation method is as follows: (1) Enhancement of fatty acid supply ability; (2) Enhancement of amino acid supply capacity; (3) Enhanced ATP supply; and (4) Deficiency or weakening of N-acylamino acid degrading enzyme The method can be expressed as a method for producing an N-acyl-amino group-containing compound in which an amino group-containing compound and a carboxyl group-containing compound are linked by an amide bond, the method comprising culturing, in the presence of a carbon source, a microorganism that has been genetically modified in at least one way selected from the group consisting of the above, and further comprising an expression unit of a polynucleotide encoding an enzyme having N-acylation activity. The details of (1) to (4) above are the same as those described above (other parts also apply).

[0105] Preferably, the direct fermentation method comprises the steps of: (a) Acyl-CoA synthetase deficiency or weakening; and (b) Acyl-ACP thioesterase enhancement The method can be expressed as a method for producing an N-acyl-amino group-containing compound in which an amino group-containing compound and a carboxyl group-containing compound are linked by an amide bond, the method comprising culturing, in the presence of a carbon source, a microorganism that has been genetically modified with at least one gene selected from the group consisting of the above, and further comprising an expression unit of a polynucleotide encoding an enzyme having N-acylation activity. The details of (a) and (b) above are the same as those described above (other parts also apply).

[0106] Examples of carbon sources include sugars, lipids, proteins, and alcohols (e.g., glycerin), with sugars being preferred. Examples of sugars include glucose, galactose, mannose, fructose, sucrose, maltose, lactose, starch hydrolysates, and molasses, with glucose being preferred.

[0107] The details of the genetic modifications (a) and (b) are as described above. At least one genetic modification selected from (a) and (b) suppresses the loss of substrates for enzymatic reactions and / or promotes their supply, thereby enhancing the metabolism of carbon sources into carboxyl group-containing compounds (e.g., fatty acids) in microorganisms.

[0108] An amino group-containing compound (e.g., amino acid) and a carboxyl group-containing compound (e.g., fatty acid) produced by metabolism are amide-bonded by the action of an enzyme with N-acylation activity, resulting in the production of an N-acyl-amino group-containing compound (e.g., N-acyl amino acid) as the desired product.

[0109] The term "enzyme with N-acylation activity" refers to an enzyme that has the ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner. Examples of enzymes with N-acylation activity include the modified enzymes with N-acylation activity described above (modified enzymes of the present invention), proteins that have been found to have the ability to form an amide bond by binding a carboxyl group and an amino group in an ATP-dependent manner, proteins that contain an amino acid sequence of the protein that includes substitution, deletion, insertion, or addition of one or several amino acids and have N-acylation activity, and proteins that contain an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein and have N-acylation activity. Proteins that have been found to have the ability to combine a carboxyl group and an amino group in an ATP-dependent manner to form an amide bond include, for example, the GH3 proteins (AtGH3-6, OsGH3-8, AtJAR1 (AtGH3-11), AtGH3-5, AtGH3-10, AtGH3-12, AtGH3-17, SsGH3, and CfHP (WP_002626336)) and PaaK proteins (PsIAAL and PaHP (WP_031591948)) described in PCT / JP2019 / 007681, and preferably AtGH3-6 (SEQ ID NO: 1). GH3 proteins and PaaK proteins also include their mutants (proteins that have an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of the protein and that have N-acylation activity, as well as proteins that have an amino acid sequence that is 90% or more identical to the amino acid sequence of the protein and that have N-acylation activity).

[0110] Examples of amino group-containing compounds produced by metabolism include those listed above as examples of amino group-containing compounds, with amino acids being preferred, and L-glutamic acid or L-aspartic acid being more preferred. Examples of carboxyl group-containing compounds produced by metabolism include those listed above as examples of carboxyl group-containing compounds, with fatty acids (e.g., saturated fatty acids) being preferred, fatty acids having 8 to 18 carbon atoms (e.g., saturated fatty acids) being more preferred, fatty acids having 12 carbon atoms being even more preferred, and lauric acid being the most preferred.

[0111] When an N-acyl-amino group-containing compound is produced by culturing a transformed microorganism, the culture conditions for the transformed microorganism are not particularly limited. For example, the culture conditions described above can be used in which the transformed microorganism is cultured in a medium further containing a predetermined amount of a carbon source (e.g., a sugar such as glucose).

[0112] 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., 1.4% (w / v) phosphoric acid, 75% (v / v) aqueous methanol solution) to the reaction system, centrifuging the mixture, and then analyzing the supernatant with UPLC-MS.

[0113] (surfactant) The N-acyl-amino group-containing compound (e.g., the N-acyl-amino group-containing compound produced by the method of the present invention) or its salt may be used in various applications, for example, as a component contained in a composition such as a cosmetic ingredient (e.g., a surfactant). The N-acyl-amino group-containing compound or its salt contained in such a composition may, for example, include an N-monounsaturated acyl-amino group-containing compound or its salt. Examples of the N-monounsaturated acyl-amino group-containing compound or its salt include those described above.

[0114] When an N-acyl-amino group-containing compound or its salt is used as a surfactant component, the surfactant may contain an N-monounsaturated acyl-amino group-containing compound or its salt having a monounsaturated acyl group having 10 to 16 carbon atoms. In such cases, since the acyl moiety of the N-monounsaturated acyl-amino group-containing compound is monounsaturated acyl, an increase in viscosity is suppressed, and the surfactant has the effects of having a low viscosity that is easy to handle and good solubility. Furthermore, since the acyl moiety has 10 to 16 carbon atoms, the surfactant provides good foaming and foam quality.

[0115] Examples of salts of N-acyl-amino group-containing compounds include those mentioned above.

[0116] When the N-acyl-amino group-containing compound is obtained from a monounsaturated fatty acid, the N-acyl-amino group-containing compound can be, for example, C n-1 H m The monounsaturated fatty acid derivative represented by -COX (X is any monovalent group, for example, a halogen atom selected from the group consisting of fluorine, chlorine, bromine, and iodine) may be obtained by reacting with a salt of an acidic amino acid (examples of the salt include the inorganic salts and organic salts described above).

[0117] Monounsaturated fatty acids from which the N-monounsaturated acyl-amino group-containing compounds are derived include, for example, decenoic acid (C10:1), dodecenoic acid (C12:1), tetradecenoic acid (C14:1) (e.g., myristoleic acid), and hexadecenoic acid (C16:1) (e.g., palmitoleic acid), with dodecenoic acid (C12:1) or hexadecenoic acid (C16:1) being preferred.

[0118] The "acidic amino acid" in the "N-acyl-amino group-containing compound" refers to an amino acid having an acidic side chain. Examples of the acidic amino acid include glutamic acid and aspartic acid, with glutamic acid being preferred.

[0119] Examples of N-monounsaturated acyl-amino group-containing compounds include N-decenoyl acidic amino acid (C10:1), N-dodecenoyl acidic amino acid (C12:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1), with N-dodecenoyl acidic amino acid (C12:1) or N-hexadecenoyl acidic amino acid (C16:1) being preferred, N-dodecenoyl glutamic acid (C12:1) or N-hexadecenoyl glutamic acid (C16:1) being more preferred, and N-dodecenoyl glutamic acid (C12:1) being even more preferred.

[0120] The N-monounsaturated acyl-amino group-containing compound may be one type of N-monounsaturated acyl-amino group-containing compound, or may contain two or more types of N-monounsaturated acyl-amino group-containing compounds. That is, the N-monounsaturated acyl-amino group-containing compound may contain one or more amino acids selected from the group consisting of N-decenoyl acidic amino acid (C10:1), N-dodecenoyl acidic amino acid (C12:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1). When two or more types of N-monounsaturated acyl-amino group-containing compounds are used, a combination of N-dodecenoyl acidic amino acid (C12:1) with one or more selected from the group consisting of N-decenoyl acidic amino acid (C10:1), N-tetradecenoyl acidic amino acid (C14:1), and N-hexadecenoyl acidic amino acid (C16:1) is preferred, and from the viewpoint of improving foam quality, a combination of N-dodecenoyl acidic amino acid (C12:1) with N-hexadecenoyl acidic amino acid (C16:1) is more preferred.

[0121] The unsaturated bond site of the N-monounsaturated acyl acidic amino acid is not particularly limited, and may be at the carbon chain terminal of the acyl moiety, between carbon atoms several carbon atoms away from the carbon chain terminal of the acyl moiety, or the α-position of the carbonyl group of the acyl moiety. Among these, it is preferable for the unsaturated bond to be between carbon atoms 6 to 7 carbon atoms away from the carbon chain terminal of the acyl moiety. That is, the N-monounsaturated acyl acidic amino acid is preferably a compound represented by the following general formula (1):

[0122] [ka] (In general formula (1), l is an integer of 1 or 2, and m is an integer of 0 to 6.)

[0123] The stereochemistry (unsaturated double bond site) of the N-monounsaturated acyl acidic amino acid may be either cis or trans, although cis is preferred from the viewpoint of suppressing an increase in viscosity and achieving a low viscosity that is easy to handle.

[0124] (composition) The composition of the present invention comprises component (A) an N-monounsaturated acyl acidic amino acid (wherein the unsaturated acyl is an acyl having 10 to 16 carbon atoms) or a salt thereof, and component (B) an N-saturated acyl acidic amino acid or a salt thereof. The composition of the present invention contains component (A), and therefore has good foaming, foam quality, and solubility even in the weakly acidic pH range similar to that of skin, and has low viscosity for easy handling. Furthermore, the composition of the present invention also contains component (B), which results in a composition with a variety of acyl groups, and therefore has the effect of improving the cleansing power for oils.

[0125] (Component (A)) Details of component (A) are as described above in the description of the N-monounsaturated acyl acidic amino acid surfactant.

[0126] (Component (B)) As used herein, the term "N-saturated acyl acidic amino acid" refers to a Cn-1 H m -CO-NH-CHR-COOH (H m The hydrogen atom represented by may be substituted. m is as defined above. R represents the side chain of an acidic amino acid. ) That is, an "N-saturated acyl acidic amino acid" refers to a compound in which one hydrogen atom on the amino group of an acidic amino acid is substituted with a saturated acyl group. An "N-saturated acyl acidic amino acid" may be a free form of an N-saturated acyl acidic amino acid, or a salt of an N-saturated acyl acidic amino acid. The salts of N-saturated acyl acidic amino acids are similar to the salts of N-monounsaturated acyl acidic amino acids.

[0127] As used herein, N-saturated acyl acidic amino acids are conveniently referred to as C n-1 H m In this specification, "saturated acyl having n carbon atoms" and the fatty acids derived therefrom are sometimes expressed as "Cn." For example, "N-lauroyl acidic amino acid (C12)" refers to a compound (C12) in which one hydrogen atom on the amino group of an acidic amino acid is substituted with a lauroyl group, which is an acyl group derived from lauric acid (C12). 11 H 23 CO—NH—CHR—COOH, where R represents the side chain of an acidic amino acid.

[0128] When the N-saturated acyl acidic amino acid is obtained from a saturated fatty acid, the N-saturated acyl acidic amino acid may be, for example, C n-1 H m The saturated fatty acid derivative represented by -COX (X is any monovalent group, for example, a halogen atom selected from the group consisting of fluorine, chlorine, bromine, and iodine) can be obtained by reacting an acidic amino acid or a salt thereof (examples of the salt include the inorganic salts and organic salts described above).

[0129] Examples of N-saturated acyl acidic amino acids include N-capryloyl acidic amino acid (C8), N-caproyl acidic amino acid (C10), N-lauroyl acidic amino acid (C12), N-myristoyl acidic amino acid (C14), N-palmitoyl acidic amino acid (C16), and N-stearoyl acidic amino acid (C18).

[0130] The "acidic amino acid" in "N-saturated acyl acidic amino acid" refers to an amino acid having an acidic side chain. Examples of acidic amino acids include glutamic acid and aspartic acid, with glutamic acid being preferred.

[0131] The N-saturated acyl acidic amino acid is preferably N-capryloyl acidic amino acid (C8), N-caproyl acidic amino acid (C10), N-lauroyl acidic amino acid (C12), N-myristoyl acidic amino acid (C14), or N-palmitoyl acidic amino acid (C16), more preferably N-lauroyl acidic amino acid (C12), and even more preferably N-lauroyl glutamic acid (C12).

[0132] Component (B) may be one type of N-saturated acyl acidic amino acid or a salt thereof, or may contain two or more types of N-saturated acyl acidic amino acids or salts thereof. That is, component (B) may contain one or more amino acids selected from the group consisting of N-capryloyl acidic amino acid (C8), N-caproyl acidic amino acid (C10), N-lauroyl acidic amino acid (C12), N-myristoyl acidic amino acid (C14), N-palmitoyl acidic amino acid (C16), and N-stearoyl acidic amino acid (C18). When there are two or more types of N-saturated acyl acidic amino acids, it is preferable to include N-lauroyl acidic amino acid (C12), and more preferably to include a combination of N-lauroyl acidic amino acid (C12) with one or more selected from the group consisting of N-capryloyl acidic amino acid (C8), N-caproyl acidic amino acid (C10), N-myristoyl acidic amino acid (C14), and N-palmitoyl acidic amino acid (C16).

[0133] When component (B) contains an N-lauroyl acidic amino acid (C12), the content of the N-lauroyl acidic amino acid (C12) in component (B) is, for example, 30% by mass or more, preferably 40% by mass or more, more preferably 50% by mass or more, and even more preferably 60% by mass or more. More specifically, the content of the N-lauroyl acidic amino acid in component (B) is, for example, 30 to 100% by mass, preferably 40 to 100% by mass, more preferably 50 to 100% by mass, and even more preferably 60 to 100% by mass.

[0134] The mass ratio of component (A) to the total of component (A) and component (B) (A / (A+B)) is usually 0.001 or more, preferably 0.002 or more, more preferably 0.003 or more, and even more preferably 0.004 or more, from the viewpoint of improving solubility at low pH by component (A). Also, from the viewpoint of contributing to the oil detergency due to the high content of component (B), the mass ratio of component (A) to the total of component (A) and component (B) (A / (A+B)) is usually 1.00 or less, preferably less than 1.00, more preferably 0.80 or less, even more preferably 0.60 or less, and even more preferably 0.50 or less. More specifically, the mass ratio of component (A) to the total of component (A) and component (B) (A / (A+B)) is usually 0.001 to 1.00, preferably 0.001 or more and less than 1.00, more preferably 0.002 to 0.80, even more preferably 0.003 to 0.60, and even more preferably 0.004 to 0.50.

[0135] The composition of the present invention may further comprise component (C) an N-unsaturated fatty acid or a salt thereof. When the composition of the present invention contains component (C), the foam quality is improved and the oil detergency is further improved.

[0136] (Component (C)) The number of carbon atoms in the unsaturated fatty acid is preferably 6 to 22, and more preferably 8 to 18. Examples of unsaturated fatty acids include hexenoic acid (C6:1), octenoic acid (C8:1), decenoic acid (C10:1), dodecenoic acid (C12:1), tetradecenoic acid (C14:1) (e.g., myristoleic acid), hexadecenoic acid (C16:1) (e.g., palmitoleic acid), octadecenoic acid (C18:1) (e.g., oleic acid), icosenoic acid (C20:1) (e.g., eicosenoic acid), and docosenoic acid (C22:1), with dodecenoic acid (C12:1) being preferred.

[0137] Examples of salts of unsaturated fatty acids include inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts; organic amine salts such as ammonium salts, monoethanolamine salts, diethanolamine salts, and triethanolamine salts; and organic salts such as basic amino acid salts such as arginine salts and lysine salts. Of these, triethanolamine salts, sodium salts, and potassium salts are preferred.

[0138] Component (C) may be one type of unsaturated fatty acid or its salt, or may contain two or more types of unsaturated fatty acids or their salts. That is, component (C) may contain one or more selected from the group consisting of hexenoic acid (C6:1), octenoic acid (C8:1), decenoic acid (C10:1), dodecenoic acid (C12:1), tetradecenoic acid (C14:1) (e.g., myristoleic acid), hexadecenoic acid (C16:1) (e.g., palmitoleic acid), octadecenoic acid (C18:1) (e.g., oleic acid), icosenoic acid (C20:1) (e.g., eicosenoic acid), and docosenoic acid (C22:1). When two or more types of unsaturated fatty acids or salts thereof are used, it is preferable that dodecenoic acid (C12:1) is included, and it is more preferable that dodecenoic acid (C12:1) is included in combination with one or more selected from the group consisting of octenoic acid (C8:1), decenoic acid (C10:1), tetradecenoic acid (C14:1), and hexadecenoic acid (C16:1).

[0139] When component (C) contains dodecenoic acid (C12:1), the content of dodecenoic acid (C12:1) in component (C) is, for example, 30% by mass or more, preferably 40% by mass or more, more preferably 50% by mass or more, even more preferably 60% by mass or more, even more preferably 70% by mass or more, and particularly preferably 80% by mass or more. More specifically, the content of dodecenoic acid (C12:1) in component (C) is, for example, 30 to 100% by mass, preferably 40 to 100% by mass, more preferably 50 to 100% by mass, even more preferably 60 to 100% by mass, even more preferably 70 to 100% by mass, and particularly preferably 80 to 100% by mass.

[0140] The mass ratio (C / (A+B)) of component (C) to the total of components (A) and (B) may be 0 or greater. When the mass ratio (C / (A+B)) of component (C) to the total of components (A) and (B) is 0, component (C) is not included. From the viewpoint of the contribution of component (C) to improving foam quality (density), the mass ratio can be 0.01 or greater, preferably 0.1 or greater, more preferably 0.2 or greater, even more preferably 0.4 or greater, and even more preferably 0.5 or greater. From the viewpoint of the contribution of the effects of components (A) and (B), the mass ratio (C / (A+B)) of component (C) to the total of components (A) and (B) is also usually 20 or less, preferably 15 or less, more preferably 13 or less, even more preferably 11 or less, and even more preferably 10 or less. More specifically, the mass ratio of component (C) to the total of component (A) and component (B) (C / (A+B)) is usually 0.01 to 20, preferably 0.1 to 15, more preferably 0.2 to 13, even more preferably 0.4 to 11, and even more preferably 0.5 to 10.

[0141] The composition of the present invention may contain component (A), component (B), and optional component (C) in (D) an aqueous medium. Any aqueous solvent can be used as the aqueous medium. Examples of the aqueous medium include aqueous solutions. The aqueous solution may or may not have buffering capacity. Examples of aqueous solutions include water (e.g., distilled water, sterilized distilled water, purified water, physiological saline, tap water such as city water), phosphate buffer, Tris-hydrochloric acid buffer, TE (Tris-EDTA) buffer, carbonate buffer, borate buffer, tartrate buffer, glycine buffer, citrate buffer, and acetate buffer.

[0142] The content of component (A) is not particularly limited, as it varies depending on various conditions, such as the type and concentration of other components contained in the composition of the present invention, and pH. When the composition of the present invention is in the form of an aqueous solution, the content is, for example, 0.001% by mass or more, preferably 0.002% by mass or more, and more preferably 0.003% by mass or more, from the viewpoint of improving solubility at low pH. The content of component (A) can also be, for example, 60% by mass or less, preferably 40% by mass or less. More specifically, the content of component (A) is, for example, 0.001 to 60% by mass, preferably 0.001 to 40% by mass, more preferably 0.002 to 40% by mass, and even more preferably 0.003 to 40% by mass.

[0143] The content of component (B) is not particularly limited, as it varies depending on various conditions, such as the type and concentration of other components contained in the composition of the present invention, as well as pH. When the composition of the present invention is in the form of an aqueous solution, the content is, for example, 0.01% by mass or more, preferably 0.02% by mass or more, and more preferably 0.03% by mass or more, from the viewpoint of improving solubility at low pH. The content of component (B) can also be, for example, 60% by mass or less, preferably 40% by mass or less, and more preferably 10% by mass or less. More specifically, the content of component (B) is, for example, 0.01 to 60% by mass, preferably 0.02 to 40% by mass, and more preferably 0.03 to 10% by mass.

[0144] The content of component (C) is not particularly limited, as it varies depending on various conditions such as the type and concentration of other components contained in the composition of the present invention, as well as pH, but may be 0% by mass or more. When the content of component (C) is 0% by mass, component (C) is not contained. When the composition of the present invention is in the form of an aqueous solution, from the viewpoint of contributing to improving foam quality (density), the content is, for example, 0.001% by mass or more, preferably 0.002% by mass or more, and more preferably 0.003% by mass or more. Furthermore, from the viewpoint that a large amount of component (C) inhibits the effects of the present invention, the content can be, for example, 5% by mass or less, preferably 3% by mass or less, and more preferably 1% by mass or less. More specifically, the content of component (C) is, for example, 0.001 to 5% by mass, preferably 0.002 to 3% by mass, and more preferably 0.003 to 1% by mass.

[0145] The composition of the present invention is preferably weakly acidic from the viewpoints of storage stability due to the inhibition of bacterial growth (antiseptic effect) and low skin irritation due to a pH equivalent to that of skin (weakly acidic). The pH of the composition of the present invention is, for example, 3.0 to 9.0, and can be 3.0 to 8.0 or 3.0 to 7.0. The upper limit of the pH is preferably 8.0 or less, more preferably 7.0 or less. The lower limit is preferably 4.0 or more, more preferably 4.5 or more. From the viewpoint of achieving a pH equivalent to that of skin, the pH of the composition of the present invention is preferably 4.0 to 8.0, more preferably 4.0 to 7.0, or preferably 4.5 to 8.0, more preferably 4.5 to 7.0. The composition of the present invention has excellent solubility even at low pH, thereby inhibiting precipitation, and in this respect, it can be said to have excellent storage stability. Furthermore, while anionic surfactants generally tend to foam less as the pH decreases, the composition of the present invention exhibits good foaming even at low pH. The pH can be adjusted using a pH adjuster. Examples of pH adjusters include the above-mentioned aqueous solutions (buffer solutions), acidic substances (e.g., hydrochloric acid, sulfuric acid, nitric acid, citric acid), and alkaline substances (e.g., hydroxides of alkali metals such as sodium and potassium, and alkaline earth metals such as calcium).

[0146] From the viewpoint of handleability, the viscosity of the composition of the present invention is preferably low. The viscosity of the composition of the present invention is, for example, 1.5 Pa·s or less, preferably 1.0 Pa·s or less, more preferably 0.80 Pa·s or less, even more preferably 0.70 Pa·s or less, and even more preferably 0.50 Pa·s or less. Because the composition of the present invention has excellent handleability, it can be said to be useful as a cosmetic for personal care. In this specification, the viscosity is a value measured by using a Brookfield viscometer (DVB-10 Brookfield viscometer manufactured by Toyo Seiki Seisakusho, rotor No. 20-23, 6-30 rpm, 25°C, 30 seconds later) to measure an aqueous solution of the composition having a concentration of 10% by mass. The composition of the present invention may also contain other ingredients such as additional cleansing ingredients, polyhydric alcohols, thickeners, stabilizers, preservatives, fragrances, colorants, etc. The specific types and amounts of these ingredients can be determined as appropriate.

[0147] Examples of additional cleansing components include surfactants such as anionic surfactants, amphoteric surfactants, and nonionic surfactants, and fine solids (eg, microspheres, scrubs).

[0148] The anionic surfactant contains one or more anionic groups. Examples of the anionic group include a carboxyl group, a sulfonic acid group, a sulfate group, and a phosphate group. Examples of the anionic surfactant include higher fatty acids, N-acyl amino acids, N-acyltaurines, alkyl ether carboxylic acids, alkyl phosphates, polyoxyethylene alkyl ether phosphates, alkyl sulfates, polyoxyethylene alkyl ether sulfates, sulfonic acid compounds having alkyl chains, and salts thereof.

[0149] The amphoteric surfactant contains one or more anionic groups as described above and one or more cationic groups. Examples of the cationic group include an ammonium group, a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary amino group. Examples of the amphoteric surfactant include an amidobetaine amphoteric surfactant, an acetate betaine amphoteric surfactant, a sulfobetaine amphoteric surfactant, and an imidazoline amphoteric surfactant (e.g., lauroamphoacetic acid or a salt thereof).

[0150] Examples of nonionic surfactants include ester-type surfactants such as glycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters; ether-type surfactants such as alkyl polyethylene glycols and polyoxyethylene alkyl phenyl ethers; and alkyl glycosides and alkyl polyglycosides in which a sugar and a higher alcohol are glycosidicly bonded.

[0151] Examples of polyhydric alcohols include dihydric alcohols (e.g., ethylene glycol, propylene glycol, 1,3-propanediol, 1,2-butylene glycol, 1,3-butylene glycol, 2,3-butylene glycol, 1,4-butanediol, 2-butene-1,4-diol, 1,5-pentanediol, 1,2-pentanediol, isoprene glycol, hexylene glycol, diethylene glycol, dipropylene glycol, monoglycerides (monoacylglycerols)), trihydric alcohols (e.g., glycerin, trimethylolpropane, 1,2,6-hexanetriol), tetrahydric alcohols (e.g., diglycerin, pentaerythritol), higher hydric alcohols, and salts thereof (e.g., the inorganic salts and organic salts described above). Examples of higher hydric alcohols include optionally substituted sugar alcohols (e.g., monosaccharide alcohols such as sorbitol, mannitol, sucrose, glucose, and mannose, disaccharide alcohols such as trehalose, and polysaccharide alcohols such as hyaluronic acid and xanthan gum), as well as polymers of the above-mentioned dihydric to tetrahydric alcohols (e.g., polyglycols and polyglycerins), and salts thereof (e.g., the above-mentioned inorganic salts and organic salts). The polyhydric alcohol is preferably a dihydric to tetrahydric alcohol, and more preferably a dihydric or trihydric alcohol.

[0152] Examples of thickeners include carrageenan, dextrin, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyacrylic acid, polymethacrylic acid, carboxyvinyl polymer (carbomer), (acrylic acid / C10-30 alkyl acrylate) copolymer, and xanthan gum.

[0153] Stabilizers include, for example, ascorbic acid, sodium pyrosulfite, and EDTA.

[0154] Examples of preservatives include ethyl parahydroxybenzoate, sodium benzoate, salicylic acid, sorbic acid, parabens (methylparaben, propylparaben, etc.), and sodium hydrogen sulfite.

[0155] The fragrances include, for example, natural fragrances and synthetic fragrances. Examples of natural fragrances include rose oil, jasmine oil, neroli oil, lavender oil, ylang-ylang oil, tuberose oil, clary sage oil, clove oil, peppermint oil, geranium oil, patchouli oil, sandalwood oil, cinnamon oil, coriander oil, nutmeg oil, pepper oil, lemon oil, orange oil, bergamot oil, opoponax oil, vetiver oil, orris oil, and oakmoss oil. Examples of synthetic fragrances include limonene (orange), β-caryophyllene (woody), cis-3-hexenol (fresh green young leaves), linalool (lily of the valley), farnesol (fresh green floral notes), β-phenylethyl alcohol (rose), 2,6-nonadienal (violet, cucumber), citral (lemon), α-hexyl cinnamic aldehyde (jasmine), β-ionone (violet when diluted), and ι-carvone (spearmint). ), cyclopentadecanone (musk), linalyl acetate (bergamot, lavender), benzyl benzoate (balsam), gamma-undecalactone (peach), eugenol (clove), rose oxide (green floral), indole (when diluted, becomes jasmine), phenylacetaldehyde dimethyl acetal (hyacinth), aurantiol (orange flower), and menthol (peppermint) (the numbers in parentheses indicate the fragrance).

[0156] Examples of pigments include organic pigments (e.g., red pigments such as Red No. 201, blue pigments such as Blue No. 404, orange pigments such as Orange No. 203, yellow pigments such as Yellow No. 205, green pigments such as Green No. 3, organic lake pigments such as zirconium lake, and natural pigments such as chlorophyll), and inorganic pigments (e.g., white pigments such as titanium oxide, colored pigments such as iron oxide, extender pigments such as talc, and pearl pigments such as mica).

[0157] The composition of the present invention can be provided in various forms, such as powder, liquid, gel, paste, cream, foam, etc. The composition of the present invention can be produced according to a conventional method.

[0158] The composition of the present invention can be made into any form of cosmetic that can be applied to, for example, skin, hair, scalp, etc., according to a conventional method. The cosmetic is suitable for use as a cleanser for animals such as humans, for example, in body shampoo, hand soap, facial cleanser, cleansing lotion, cleansing cream, massage cream, hair shampoo, etc. The preferred properties (e.g., pH) of the cosmetic are the same as those of the composition of the present invention described above. The composition of the present invention can also be used as an additive such as an excipient. [Example]

[0159] 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.

[0160] Example 1: Construction of mutant AtGH3-6 expression plasmid by site-directed mutagenesis (1) Mutations were introduced into AtGH3-6 using pET-28a-AtGH3-6 (PCT / JP2019 / 007681) as a template, which expresses Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.6 (AtGH3-6, Q9LSQ4; amino acid sequence: SEQ ID NO: 1; nucleotide sequence encoding the amino acid sequence, codon-optimized for expression in E. coli: SEQ ID NO: 2). Mutations were introduced into several mutant AtGH3-6 strains using the mutant AtGH3-6 expression plasmid obtained below as a template. The method for constructing the expression plasmid for mutant AtGH3-6 (Mutant No. 31) was described in Example 1(2). Mutations were introduced using PrimeSTAR Max DNA Polymerase (Takara Bio) under the following conditions. 1 cycle: 98℃, 30 seconds 30 cycles 98℃, 10 seconds 55℃, 15 seconds 72℃, 40 seconds 1 cycle: 72℃, 5 min 4℃, hold

[0161] The primers used are as follows: WT in the table indicates wild-type AtGH3-6.

[0162] [Table 2-1]

[0163] [Table 2-2]

[0164] [Table 2-3]

[0165] The resulting PCR product was digested with DpnI, and E. coli JM109 was transformed with the reaction mixture. The target plasmid was extracted from the kanamycin-resistant strain. This plasmid was used as the mutant AtGH3-6 expression plasmid.

[0166] (2) Mutations were introduced into AtGH3-6 using pET-28a-AtGH3-6 (PCT / JP2019 / 007681) as a template. Furthermore, for some mutant AtGH3-6, further mutations were introduced using the mutant AtGH3-6 expression plasmids obtained above or below as templates. Mutations were introduced using PrimeSTAR GXL DNA Polymerase (Takara Bio) under the following conditions. 1 cycle: 98℃, 30 seconds 30 cycles 98℃, 10 seconds 60℃, 15 seconds 68℃, 7.5 min 1 cycle: 72℃, 5 min 4℃, hold

[0167] The primers used are as follows:

[0168] [Table 3]

[0169] The resulting PCR product was digested with DpnI, and the reaction mixture was used to transform E. coli JM109. The target plasmid was extracted from the kanamycin-resistant strain. This plasmid was designated the mutant AtGH3-6 expression plasmid. Sequence confirmation revealed that mutant 26 also contained the M337A mutation in addition to the introduced mutation site.

[0170] (3) Using pET-28a-mutant AtGH3-6 (mutant number 56) as a template, further mutations were introduced into AtGH3-6. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) under the following conditions: 1 cycle: 98℃, 30 seconds 30 cycles 98℃, 10 seconds 60℃, 15 seconds 72℃, 5sec / kb 1 cycle: 72℃, 5 min 4℃, hold

[0171] The primers used are as follows:

[0172] [Table 4]

[0173] The resulting PCR products were separated by agarose gel electrophoresis, and DNA of the desired size was extracted from the agarose gel. An In-Fusion reaction was then performed using the In-Fusion® HD Cloning Kit (Takara Bio). E. coli JM109 was transformed with the reaction mixture, and the desired plasmid was extracted from the kanamycin-resistant strain. This plasmid was designated the mutant AtGH3-6 expression plasmid.

[0174] (4) A plasmid, pET-28a-mutant AtGH3-6 (mutant number 36), in which the gene for mutant AtGH3-6 (sequence numbers 185 and 186) was inserted into the NdeI and XhoI sites in the multiple cloning site of pET-28a(+), was purchased from Eurofins Genomics.

[0175] Example 2: Construction of mutant AtGH3-6 expression plasmid by random mutagenesis Mutations were introduced into AtGH3-6 using pET-28a-AtGH3-6 (PCT / JP2019 / 007681) as a template using the Gene Morph II Random Mutagenesis Kit (Agilent Technologies) under the following conditions. 1 cycle: 95℃, 2 min 30 cycles 95℃, 30 seconds 60℃, 30 seconds 72℃, 1.2 min 1 cycle: 72℃, 10 min 4℃, hold

[0176] The primers used are as follows:

[0177] [Table 5]

[0178] The resulting approximately 2.1 kb DNA fragment was digested with NdeI and XhoI and ligated with the pET-28a-AtGH3-6 vector DNA fragment, which had also been digested with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, and plasmids were extracted from the kanamycin-resistant strain to create a mutant AtGH3-6 expression plasmid library. The resulting plasmid library was then introduced into E. coli BL21(DE3), and transformants harboring the mutant AtGH3-6 expression plasmid were isolated from the kanamycin-resistant strain. Plasmids were extracted from the resulting transformants, and the AtGH3-6 sequence was confirmed, revealing the following amino acid substitutions:

[0179] [Table 6]

[0180] Example 3: Purification of mutant AtGH3-6 The mutant AtGH3-6 expression plasmids were each introduced into E. coli BL21(DE3), and transformants containing the plasmids were isolated from kanamycin-resistant strains. This strain was inoculated into 50 mL of LB medium containing 50 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. After the culture was completed, the cells were collected from 20 mL of culture by centrifugation and suspended in 4 mL of xTractor™ Buffer (Takara Bio). 8 μL of 5 units / μL DNase I solution and 40 μL of 100× lysozyme solution included in the xTractor™ Buffer Kit (Takara Bio) were added, mixed by inversion, and then allowed to stand at room temperature for 20 minutes. The supernatant obtained by centrifugation was loaded onto a TALON™ Spin Column (Takara Bio) and purified according to the manufacturer's protocol. The equilibration buffer was 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The elution buffer was 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 150 mM imidazole. The resulting eluate was collected and concentrated using an Amicon Ultra-0.5 10 kDa (Merck). The buffer was then exchanged to 20 mM Tris-HCl (pH 8.0), 1 mM DTT, and the purified enzyme solution was obtained. The protein concentration of the purified enzyme solution was measured using Protein Assay CBB Solution (5x concentrated) (Nacalai Tesque) and the Quick Start BSA Standard Set (Bio-Rad).

[0181] Example 4: Activity measurement of mutant AtGH3-6 for each amino acid substrate A 0.2 mL reaction mixture containing 50 mM Tris-HCl, 5 mM L-glutamic acid or L-aspartic acid, 5 mM sodium laurate, 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 stop solution (1.4% (w / v) phosphoric acid, 75% (v / v) methanol) was added, and the supernatant was centrifuged and subjected to UPLC-MS analysis. The peak area corresponding to the molecular weight signals of Nα-lauroyl-L-glutamic acid (C12-L-Glu) or Nα-lauroyl-L-aspartic acid (C12-L-Asp) was extracted using selected ion recording (SIR) to confirm the peak area. The amount of product was quantified using a calibration curve of the standard product.

[0182] The UPLC-MS analysis conditions are as follows. (Analysis conditions 1) Instrument: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 x 50 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile Gradient: A:B 60:40~20:80 Gradient 1.2min A:B 20:80~60:40 Gradient 0.01min A:B 60:40 Isocratic 0.29min Flow rate: 0.6mL / min Injection volume: 2 μL Column temperature: 40℃ Ionization method: ESI-negative SIR:C12-L-Glu m / z 328[MH] - C12-L-Asp m / z 314[M−H] -

[0183] (Analysis conditions 2) 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: A:B 60:40~20:80 Gradient 2.4min A:B 20:80~60:40 Gradient 0.1min A:B 60:40 Isocratic 0.5min Flow rate: 0.6mL / min Injection volume: 2 μL Column temperature: 40℃ Ionization method: ESI-negative SIR:C12-L-Glu m / z 328[MH] - C12-L-Asp m / z 314[M−H] -

[0184] UPLC-MS analysis confirmed that mutant AtGH3-6 had improved Nα-lauroyl-L-glutamic acid (C12-L-Glu) production and / or improved substrate specificity for L-glutamic acid (amount of C12-L-Glu produced / amount of C12-L-Asp produced) compared to wild-type AtGH3-6. WT in the table indicates wild-type AtGH3-6.

[0185] [Table 7-1]

[0186] [Table 7-2]

[0187] [Table 7-3]

[0188] [Table 7-4]

[0189] Example 5: Measurement of activity of mutant AtGH3-6 against each fatty acid substrate A 0.2 mL reaction mixture containing 50 mM Tris-HCl, 5 mM L-glutamic acid, 5 mM sodium fatty acid (or fatty acid), 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. The sodium fatty acid (or fatty acid) used in the enzymatic reaction was dissolved in 20% (v / v) Triton X-100 to prepare a 25 mM solution, which was then added to the reaction mixture to a final concentration of 5 mM. After completion of the reaction, 0.8 mL of stop solution (1.4% (w / v) phosphoric acid, 75% (v / v) methanol) was added, and the supernatant was centrifuged and subjected to UPLC-MS analysis. Signals corresponding to the predicted molecular weight of Nα-acyl-L-glutamic acid were extracted using SIR, and the peak area was confirmed. The amount of product was quantified using a calibration curve of the standard sample. C8-L-Glu was used as a standard for quantifying C6-L-Glu and C8-L-Glu, C10-L-Glu for quantifying C10-L-Glu, and C12-L-Glu for quantifying Nα-acyl-L-Glu with an acyl group of C12 or longer. The sodium fatty acids used were sodium caproate, sodium caprylate, sodium caprate, sodium laurate, sodium myristate, sodium palmitate, cis-5-dodecenoic acid, cis-9-tetradecenoic acid, or cis-9-hexadecenoic acid. Saturated fatty acids with x carbon atoms were designated Cx, and unsaturated fatty acids with x carbon atoms and y carbon-carbon double bonds were designated Cx:y.

[0190] The UPLC-MS analysis conditions are as follows. Instrument: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 × 50 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile Gradient: A:B 90:10~0:100 Gradient 1.8min A:B 0:100 Isocratic 0.6min A:B 0:100~90:10 Gradient 0.1min A:B 90:10 Isocratic 0.5min Flow rate: 0.6mL / min Injection volume: 2 μL Column temperature: 40℃ Ionization method: ESI-negative

[0191] [Table 8]

[0192] UPLC-MS analysis confirmed that mutant AtGH3-6 produced more Nα-acyl-L-glutamates with acyl groups of eight or more carbon atoms than wild-type AtGH3-6. WT in the table indicates wild-type AtGH3-6.

[0193] [Table 9]

[0194] Example 6: Construction of fatty acid producing strain (E. coli) As a strain that produces fatty acids from carbon sources such as glucose, an acyl-CoA synthetase (fadD)-deficient / acyl-ACP thioesterase-enhanced E. coli strain (E. coli ΔfadD / pMW118-Ptac-UcTEopt) was constructed according to the following procedure.

[0195] (1) Synthesis of the UcTE (Umbellularia californica acyl-ACP thioesterase) gene Acyl-ACP thioesterase is a plant-derived enzyme involved in medium-chain fatty acid synthesis. This enzyme can be used to modify the chain length of bacterial fatty acid synthesis. Previous studies have focused on medium-chain acyl-ACP thioesterase (UcTE, GenBank: M94159) from California bay (Umbellularia californica), which produces predominantly lauric acid in oilseeds, and it has been used to produce lauric acid in E. coli (Voelker and Davies, J. of Bacteriol. 1994;176(23):7320-7327). Although a transit peptide has been identified in the N-terminal region of UcTE (SEQ ID NO: 187), it has been shown that the transit peptide is not essential for UcTE enzymatic activity (Feng et al., ACS Chem Biol. 2017;12(11):2830-2836). Therefore, we decided to use a truncated gene that does not contain the transport peptide (i.e., a gene that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 3, with an initiation methionine added at amino acid residues 84 to 382). The sequence of the truncated UcTE gene, with codons optimized for expression in E. coli, is shown in SEQ ID NO: 4. We commissioned Eurofins Genomics to synthesize the UcTE gene, with codons optimized for expression in E. coli, and purchased a plasmid (pEX-K4J1-UcTEopt) in which a DNA fragment containing the gene had been inserted into the pEX-K4J1 vector.

[0196] (2) Construction of pMW118-Sce-Km plasmid To obtain a plasmid compatible with both E. coli and P. ananatis, we constructed the expression vector pMW118-Sce-Km. This plasmid contains the kan gene (kanamycin resistance marker) and the bla gene (ampicillin resistance marker). The pMW118-Sce-Km plasmid (Figure 1) was constructed based on the pMW118-placUV5-lacI plasmid (Skorokhodova et al., Biotechnologiya (Russian). 2004;5:3-21). A DNA fragment containing the kan gene (kanamycin resistance marker) encoding aminoglycoside phosphotransferase and its RBS was amplified by PCR using pUC-4K (GenBank / EMBL accession number X06404, Pharmacia) as a template (Mashko et al., Biotekhnologiya. 2001;5:3-20). The primers used were as follows:

[0197] [Table 10]

[0198] The resulting DNA fragment was inserted into the BamHI and XbaI sites of the pMW118-placUV5-lacI plasmid by ligation using T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation mixture and grown on LB agar medium containing 50 mg / L kanamycin. pMW118-Sce-Km was extracted from the kanamycin-resistant strain. The structure of the plasmid was confirmed by sequence analysis (Figure 1).

[0199] (3) Construction of pMW118-Ptac-UcTEopt plasmid Using the pEX-K4J1-UcTEopt plasmid as a template, a DNA fragment containing a truncated UcTEopt gene fused to a T7 phage-derived RBS (RBST7) and containing a restriction enzyme site was amplified by PCR using the following primers:

[0200] [Table 11]

[0201] Using chromosomal DNA containing the Ptac promoter (Katashkina et al. Molecular Biology. 2005; 39(5): 719-726) as a template, a DNA fragment containing the Ptac promoter with restriction enzyme sites added was amplified by PCR. The primers used were as follows:

[0202] [Table 12]

[0203] The resulting DNA fragment containing RBST7 and the truncated UcTEopt gene and the DNA fragment containing the Ptac promoter were treated with XbaI and ligated using T4 DNA ligase (Thermo Fisher Scientific). Using this ligation reaction mixture as a template, a DNA fragment containing Ptac-RBST7-UcTEopt (SEQ ID NO: 6) with added BglII and EcoRI sites was amplified by PCR. The primers used were as follows:

[0204] [Table 13]

[0205] The resulting DNA fragment was inserted into the BglII and EcoRI sites of pMW118-Sce-Km by ligation using T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation reaction mixture and grown on LB agar medium containing 50 mg / L kanamycin. The pMW118-Ptac-UcTEopt plasmid was extracted from the kanamycin-resistant strain. The structure of the plasmid was confirmed by sequence analysis (Figure 2).

[0206] (4) Construction of E. coli ΔfadD / pMW118-Ptac-UcTEopt (fatty acid producing strain) To construct a fatty acid-producing E. coli strain, E. coli K-12 MG1655 (F-lambda-ilvG-rfb-50rph-ATCC 47076) was used as the base strain. To block the fatty acid degradation pathway, an in-frame deletion of the fadD gene was performed. A PCR-amplified DNA fragment containing λattL-kan-λattR, which contained a 40-bp region homologous to the fadD gene, was used to perform the deletion using the λ-Red method, as previously described (Katashkina et al., BMC Mol Biol. 2009;10:34). After electroporation, the strain was grown on LB agar medium containing 50 mg / L kanamycin. The DNA fragment used to replace the fadD gene with λattL-kan-λattR was amplified by PCR using genomic DNA containing the λattL-kan-λattR cassette (Katashkina et al., BMC Mol Biol. 2009;10:34) as a template. The primers used were as follows:

[0207] [Table 14]

[0208] The resulting E. coli MG1655 ΔfadD::λattL-kan-λattR was confirmed by PCR using the following primers:

[0209] [Table 15]

[0210] The kanamycin resistance marker (kan gene) was removed from the resulting strain using a previously reported phage λInt / Xis-dependent method (Katashkina et al., BMC Mol Biol. 2009;10:34). Removal of the marker from the chromosome was confirmed by PCR. The primers used were as follows:

[0211] [Table 16]

[0212] The resulting strain (E. coli MG1655 ΔfadD::λattB) was transformed with the pMW118-Ptac-UcTEopt plasmid by electroporation to obtain E. coli ΔfadD / pMW118-Ptac-UcTEopt as a fatty acid-producing strain.

[0213] Example 7: Construction of a fatty acid producing strain (P. ananatis) A fadD-deficient / gcd-deficient / acyl-ACP thioesterase-enhanced P. ananatis strain (P. ananatis SC17(0) ΔfadD Δgcd / pMW118-PlacUV5-lacI-UcTEopt) capable of producing fatty acids from carbon sources such as glucose was constructed according to the following procedure.

[0214] (1) Construction of pMW118-PlacUV5-lacI-UcTEopt Plasmid A DNA fragment containing RBST7 and the truncated UcTEopt gene was amplified by PCR using the pMW118-Ptac-UcTEopt plasmid described in Example 6 as a template. The primers used are as follows:

[0215] [Table 17]

[0216] The resulting DNA fragment was inserted into the EcoRI and BamHI sites of the pMW118-Sce-Km plasmid by ligation using T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation reaction mixture and grown on LB agar medium containing 50 mg / L kanamycin. The pMW118-PlacUV5-lacI-UcTEopt plasmid was extracted from the kanamycin-resistant strain. The structure of the plasmid was confirmed by sequence analysis (Figure 3).

[0217] (2) Construction of P. ananatis SC17(0) ΔfadD Δgcd / pMW118-PlacUV5-lacI-UcTEopt To construct a fatty acid-producing P. ananatis strain, we used P. ananatis SC17(0) (Katashkina JI et al., BMC Mol Biol. 2009;10:34) as the base strain. To block the fatty acid degradation pathway, an in-frame deletion of the fadD gene (PAJ_1453) was performed. A PCR-amplified DNA fragment containing λattL-kan-λattR, which contained a 40-bp region homologous to the fadD gene, was used to perform the deletion using the λ-Red method, as previously described (Katashkina et al., BMC Mol Biol. 2009;10:34). After electroporation, the strain was grown on LB agar medium containing 50 mg / L kanamycin. The DNA fragment used to replace the fadD gene with λattL-kan-λattR was amplified by PCR using genomic DNA containing the λattL-kan-λattR cassette (Katashkina et al., BMC Mol Biol. 2009;10:34) as a template. The primers used were as follows:

[0218] [Table 18]

[0219] The resulting P. ananatis SC17(0) ΔfadD::λattL-kan-λattR was confirmed by PCR using the following primers:

[0220] [Table 19]

[0221] The kanamycin resistance marker (kan gene) was removed from the resulting strain using a previously reported phage λInt / Xis-dependent method (Katashkina et al., BMC Mol Biol. 2009;10:34). Removal of the marker from the chromosome was confirmed by PCR. The primers used were as follows:

[0222] [Table 20]

[0223] The resulting strain was designated P. ananatis SC17(0) ΔfadD::λattB.

[0224] Next, we performed a deletion of the gcd gene (PAJ_3473). Genomic DNA isolated from P. ananatis SC17(0) Δgcd::λattR-attLφ80-kan-attRφ80 using the Wizard Genomic DNA Purification Kit (Promega) (Katashkina et al., Biotekhnologiya. 2019;35(2):3-15) was electroporated into P. ananatis SC17(0) ΔfadD::λattB according to a previously published method (Katashkina et al., BMC Mol Biol. 2009;10:34). Introduction of the Δgcd::λattR-attLφ80-kan-attRφ80 cassette was confirmed using the following primers:

[0225] [Table 21]

[0226] The resulting strain was designated P. ananatis SC17(0) ΔfadD::λattB Δgcd::λattR-attLφ80-kan-attRφ80. The kanamycin resistance marker (kan gene) was removed from this strain using a previously published method (Andreeva et al., FEMS Microbiol Lett. 2011;318(1):55-60) with the pAH129-cat helper plasmid. Marker removal was confirmed by PCR. The primers used were as follows:

[0227] [Table 22]

[0228] The resulting strain (P. ananatis SC17(0) ΔfadD::λattB Δgcd::λattR-attBφ80) was transformed with the pMW118-PlacUV5-lacI-UcTEopt plasmid by electroporation and grown on LB agar medium containing 50 to 200 mg / L kanamycin to obtain P. ananatis SC17(0) ΔfadD Δgcd / pMW118-PlacUV5-lacI-UcTEopt as a fatty acid-producing strain.

[0229] Example 8: Synthesis of lauric acid from glucose using a fatty acid-producing strain The fatty acid-producing strains constructed in Examples 6 and 7 were inoculated onto K-medium agar medium (K-medium: LB medium supplemented with 0.5xM9 salts and 5g / L D-glucose) containing 50mg / L kanamycin and incubated at 30°C for 16 hours. The strains evaluated are as follows:

[0230] [Table 23]

[0231] The obtained bacterial cells were inoculated into 50 mL of evaluation medium and cultured with shaking at 30°C and 220 rpm using an Ultra Yield™ Flask 500 mL (THOMSON). When culturing strain number 2, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as follows:

[0232] [Table 24]

[0233] After the incubation, 1 mL of the well-mixed culture was centrifuged to obtain bacterial cells. 1 mL of 1.4% (w / v) phosphoric acid, 75% (v / v) methanol (containing 1000 ppm tridecylic acid as an internal standard) was added to the resulting mixture and mixed for 3 minutes using a vortex mixer. The supernatant obtained by centrifugation was subjected to GC and GC-MS analysis.

[0234] The GC analysis conditions are as follows: Equipment:GC-2010(SHIMADZU) Column: DB-FFAP 30 m, ID 0.25 mm, film 0.25 μm (Agilent Technologies) Injection volume: 1μL Injection method: Split 50:1 Inlet temperature: 280℃ Column oven: 190℃ (5 min) - 8℃ / min - 250℃ (7.5 min) Carrier gas: He, linear velocity: 35 cm / sec Detector: FID, 300℃

[0235] The GC-MS analysis conditions are as follows: Instrument: Network GC System (6890N, Agilent Technologies) Mass Selective Detector (5973,Agilent Technologies) Column: DB-FFAP 30 m, ID 0.25 mm, film 0.25 μm (Agilent Technologies) Injection volume: 1μL Injection method: Split 50:1 Inlet temperature: 280℃ Column oven: 190℃ (5 min) - 8℃ / min - 250℃ (7.5 min) Carrier gas: He, linear velocity: 39 cm / sec MS temperature: 230℃ (ion source), 150℃ (quadrupole) Scan range: m / z 30-350

[0236] The amounts of various fatty acids were quantified from the peak area values ​​obtained by GC analysis. Standards were prepared by dissolving the various fatty acids in 1.4% (w / v) phosphoric acid and 75% (v / v) methanol (containing 1000 ppm tridecylic acid as an internal standard). Corrections were made between samples by comparison with the peak area value of the internal standard tridecylic acid, and the amounts of various fatty acids per culture medium were calculated. Analysis confirmed the production of various fatty acids, with lauric acid as the main component, in strains 1 and 2. The various fatty acids produced were identified by their retention times in GC analysis and GC-MS analysis.

[0237] [Table 25]

[0238] Example 9: Construction of N-acylamino acid producing strains Strains capable of producing N-acylamino acids from carbon sources such as glucose were constructed by introducing wild-type AtGH3-6 or mutant AtGH3-6 expression units into the fatty acid-producing strains constructed in Examples 6 and 7 according to the following procedure.

[0239] DNA synthesis for the promoter PphoC_SDatc sequence (SEQ ID NO: 5) was requested from Thermo Fisher Scientific, and a plasmid containing the sequence inserted into the EcoRI and NdeI sites was purchased. Using this plasmid as a template, a DNA fragment containing PphoC_SDatc was amplified by PCR. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) under the following conditions: 1 cycle: 98℃, 30 seconds 30 cycles 98℃, 10 seconds 60℃, 15 seconds 72℃, 5 seconds 1 cycle: 72℃, 5 min 4℃, hold

[0240] The primers used are as follows:

[0241] [Table 26]

[0242] DNA fragments containing the wild-type or mutant AtGH3-6 gene were amplified by PCR using pET-28a-AtGH3-6 or pET-28a-AtGH3-6 ID31, pET-28a-AtGH3-6 ID49, pET-28a-AtGH3-6 ID56, or pET-28a-AtGH3-6 ID61 (ID indicates the mutant number) constructed in Example 1 as a template. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) under the following conditions. 1 cycle: 98℃, 30 seconds 30 cycles 98℃, 10 seconds 60℃, 15 seconds 72℃, 10 seconds 1 cycle: 72℃, 5 min 4℃, hold

[0243] The primers used are as follows:

[0244] [Table 27]

[0245] PCR products containing PphoC_SDatc, PCR products containing wild-type or mutant AtGH3-6 genes, and pHSG398 (Takara Bio) digested with EcoRI and SacI were each separated by agarose gel electrophoresis. DNA of the desired size was extracted from the agarose gel and subjected to In-Fusion reactions using the In-Fusion® HD Cloning Kit (Takara Bio). E. coli JM109 was transformed with the reaction mixture, and the desired plasmid (containing the PphoC_SDatc and AtGH3-6 expression units) was extracted from a chloramphenicol-resistant strain. These plasmids were designated pHSG398-PphoC-AtGH3-6 (SDatc), pHSG398-PphoC-AtGH3-6 ID31 (SDatc), pHSG398-PphoC-AtGH3-6 ID49 (SDatc), pHSG398-PphoC-AtGH3-6 ID56 (SDatc), and pHSG398-PphoC-AtGH3-6 ID61 (SDatc). The fatty acid-producing strains constructed in Examples 6 and 7 (E. coli ΔfadD / pMW118-Ptac-UcTEopt and P. ananatis ΔfadD Δgcd / pMW118-PlacUV5-lacI-UcTEopt) were transformed with the target plasmids and pHSG398 (negative control), and transformants containing the plasmids were selected from kanamycin- and chloramphenicol-resistant strains. The resulting transformants were designated N-acyl amino acid-producing strains. The strains constructed are as follows:

[0246] [Table 28]

[0247] Example 10: Synthesis of Nα-lauroyl-L-glutamic acid from glucose using an N-acylamino acid producing strain The N-acyl amino acid producing strain constructed in Example 9 was inoculated into LB agar medium containing 50 mg / L kanamycin and 25 mg / L chloramphenicol and incubated at 30°C for 16 hours. The resulting bacterial cells were inoculated into 3 mL of evaluation medium and cultured in a test tube with shaking at 30°C and 120 rpm for 48 hours. When culturing strains 9 to 14, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as follows:

[0248] [Table 29]

[0249] After incubation, 900 μL of 1.4% (w / v) phosphoric acid / 75% (v / v) methanol was added to 100 μL of well-mixed culture medium and mixed for 3 minutes using a vortex mixer. The supernatant obtained by centrifugation was diluted appropriately with 1.4% (w / v) phosphoric acid / 75% (v / v) methanol and subjected to UPLC-MS analysis. The UPLC-MS analysis conditions were as described in Example 5.

[0250] UPLC-MS analysis showed that expression of the negative control (pHSG398) and wild-type AtGH3-6 barely produced any Nα-acyl-L-glutamates, whereas expression of mutant AtGH3-6 (ID31, 49, 56, and 61) confirmed the production of various Nα-acyl-L-glutamates. In the table, "Control" refers to the negative control, and "WT" refers to the wild-type AtGH3-6.

[0251] [Table 30]

[0252] Example 11: Synthesis of Nα-lauroyl-L-aspartic acid from glucose using an N-acylamino acid producing strain The N-acylamino acid-producing strain constructed in Example 9 was inoculated into LB agar medium containing 50 mg / L kanamycin and 25 mg / L chloramphenicol and incubated at 30°C for 16 hours. The resulting cells were inoculated into 3 mL of evaluation medium and cultured in a test tube with shaking at 30°C and 120 rpm for 48 hours. When culturing strains 9 to 14, 1 mM IPTG was added to the medium. The evaluation medium had the composition described in Example 10 supplemented with 10 g / L L-aspartic acid.

[0253] After incubation, 900 μL of 1.4% (w / v) phosphoric acid / 75% (v / v) methanol was added to 100 μL of well-mixed culture medium and mixed for 3 minutes using a vortex mixer. The supernatant obtained by centrifugation was diluted appropriately with 1.4% (w / v) phosphoric acid / 75% (v / v) methanol and subjected to UPLC-MS analysis. UPLC-MS analysis was performed as described in Example 5, and SIR detection was performed as follows. C10-L-Asp was used as a standard for quantification of C10-L-Asp, and C12-L-Asp was used for quantification of Nα-acyl-L-Asp with an acyl group of C12 or longer.

[0254] [Table 31]

[0255] UPLC-MS analysis showed that the negative control (pHSG398) barely produced any of the Nα-acyl-L-aspartic acids, but that the expression of wild-type AtGH3-6 or mutant AtGH3-6 (ID31, 49, 56, and 61) confirmed the production of various Nα-acyl-L-aspartic acids. In the table, "Control" refers to the negative control, and "WT" refers to the wild-type AtGH3-6.

[0256] [Table 32]

[0257] Example 12: Evaluation of foaming of culture medium of N-acyl amino acid producing strain The N-acyl amino acid producing strain constructed in Example 9 was inoculated onto an LB agar medium containing 50 mg / L kanamycin and 25 mg / L chloramphenicol and incubated at 30° C. for 16 hours. The strains evaluated are as follows:

[0258] [Table 33]

[0259] The obtained bacterial cells were inoculated into 50 mL of evaluation medium and cultured with shaking at 30°C and 220 rpm using an Ultra Yield™ Flask 500 mL (THOMSON). When culturing strains 9, 10, and 14, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as described in Example 10.

[0260] After incubation, the well-mixed culture medium was centrifuged. 100 μL of the resulting culture supernatant was mixed with 900 μL of 1.4% (w / v) phosphoric acid and 75% (v / v) methanol and mixed for 3 minutes using a vortex mixer. The supernatant obtained by centrifugation was diluted appropriately with 1.4% (w / v) phosphoric acid and 75% (v / v) methanol and subjected to UPLC-MS analysis. The UPLC-MS analysis conditions were as described in Example 5.

[0261] UPLC-MS analysis showed that expression of the negative control (pHSG398) and wild-type AtGH3-6 resulted in little production of various Nα-acyl-L-glutamates, whereas expression of mutant AtGH3-6 (ID61) confirmed the production of various Nα-acyl-L-glutamates. In the table, "Control" refers to the negative control, "WT" refers to the wild-type AtGH3-6, and "total" refers to the total concentration of Nα-acyl-L-glutamates with acyl groups of C10, C12, C12:1, C14, C14:1, C16, and C16:1.

[0262] [Table 34]

[0263] To evaluate the foaming of the resulting culture supernatants, the pH of 4 mL of each culture supernatant was adjusted to 5.0 with HCl, and then ultrapure water was added to make up to 5 mL. As a control, a solution was prepared by dissolving Nα-lauroyl-L-glutamic acid (Amisoft® LA-D) in ultrapure water and adjusting the pH to 5.0 with NaOH. Furthermore, a solution of Nα-lauroyl-L-glutamic acid (Amisoft® LA-D) was added to 4 mL of culture supernatant from the negative control (pHSG398), and the pH was adjusted to 5.0 with HCl. Ultrapure water was then added to make up to 5 mL.

[0264] 2 mL of each sample was placed in a screw-cap test tube (total length 105 mm, inner diameter of the mouth φ10.0 mm, body diameter φ16.5 mm) and incubated in a water bath at 35°C for 10 minutes. After that, the screw-cap test tube was held in the hand and shaken up and down for 10 seconds, and the height of the bubbles immediately afterwards was measured.

[0265] The results showed that expression of mutant AtGH3-6 (ID61) produced stronger foam than expression of the negative control (pHSG398) or wild-type AtGH3-6. Therefore, the N-acyl-L-glutamic acid produced by the N-acyl amino acid-producing strain was suitable for use as a surfactant. In the table, "Control" refers to the negative control, "WT" refers to wild-type AtGH3-6, and "Total N-acyl-L-Glu" refers to the total concentration of N-acyl-L-glutamic acid with acyl groups of C10, C12, C12:1, C14, C14:1, C16, and C16:1.

[0266] [Table 35] [Industrial Applicability]

[0267] 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]

[0268] SEQ ID NOs: 1 and 2 show the amino acid sequence of AtGH3-6 and the nucleotide sequence encoding the amino acid sequence with codons optimized for expression in Escherichia coli, respectively. SEQ ID NOs: 3 and 4 show the amino acid sequence of medium-chain acyl-ACP thioesterase (UcTE, GenBank: M94159) derived from California bay (Umbellularia californica) (amino acids 1 to 83 represent the transit peptide) and the nucleotide sequence encoding the amino acid sequence with an initiation methionine added at amino acid residues 84 to 382 of the amino acid sequence (codons optimized for expression in Escherichia coli), respectively. SEQ ID NO: 5 shows the base sequence of PphoC_SDatc. SEQ ID NO: 6 shows the base sequence of Ptac-RBST7-UcTEopt (nucleotides 7 to 71 represent the Ptac promoter, nucleotides 82 to 110 represent RBST7, and nucleotides 117 to 1019 represent the UcTEopt gene). SEQ ID NOs: 7 to 184 show the nucleotide sequences of the primers. SEQ ID NOs: 185 and 186 show the amino acid sequence of mutant AtGH3-6 and the nucleotide sequence encoding the amino acid sequence with codons optimized for expression in Escherichia coli, respectively. SEQ ID NO: 187 shows the amino acid sequence of the transit peptide of UcTE.

Claims

[Claim 1] the below described: (A) a modified amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1, including mutations of one or more amino acid residues selected from the group consisting of N101, R117, T122, I123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576; (B) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence containing one or more additional mutations of amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence; Including, A modified enzyme having N-acylation activity in which any one of the following properties (i) to (iii) is improved compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1: (i) N-acylation activity towards L-glutamic acid and / or L-aspartic acid; (ii) substrate specificity for L-glutamic acid; or (iii) N-acylation activity for L-glutamic acid and / or L-aspartic acid, and substrate specificity for L-glutamic acid.

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