Modified indole-3-acetic acid-amido synthetase GH3.6 enzyme having n-acylation activity

Abstract

The present invention provides an enzyme useful for establishing an excellent N-acyl-amino group-containing compound production system, and the like. More specifically, the present invention provides a modified enzyme comprising: (A) a modified amino acid sequence consisting of an amino acid sequence comprising mutations of one or more certain amino acid residues in an amino acid sequence of a wild type enzyme having an N-acylation activity; (B) an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence comprising additional mutations of one or more amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence, having an N-acylation activity, and having an improved N-acylation activity to L-glutamic acid or L-aspartic acid or an improved substrate specificity to L-glutamic acid as compared with the wild type enzyme, and the like.

Claims

1. A modified enzyme comprising a mutation selected from the group consisting of N101S, I123T, Y134F, Y134V, L137I, V140I, S161P, V174A, Q200E, V231A, V311A, T336S, M337G, A339G, S340A, Y344A, Y344G, Y344V, K388N, E483D, C576A and combinations thereof in an amino acid sequence selected from the group consisting of: (A) the amino acid sequence of SEQ ID NO: 1; (B) the amino acid sequence of (A), but comprising a further mutation comprising substitution, deletion, insertion, or addition of one to thirty amino acid residues; and (C) the amino acid sequence of (A), but comprising additional mutations of one or more amino acid residues and having 95% or more identity to the amino acid sequence of (A), and wherein said modified enzyme has an N-acylation activity comprising: (i) an N-acylation activity to L-glutamic acid and/or L-aspartic acid; and/or (ii) a substrate specificity to L-glutamic acid; wherein the N-acylation activity is improved over a non-modified enzyme consisting of the amino acid sequence of SEQ ID NO: 1.

2. The modified enzyme according to claim 1, further comprising an additional mutation selected from the group consisting of R117P, T122S, C335S, M337A, Y344I, R350T, G379D, L390P, S455T, Q533R, and combinations thereof.

3. The modified enzyme according to claim 1, wherein the N-acylation activity is to L-glutamic acid or L-aspartic acid.

4. A method for producing an N-acyl-amino group-containing compound or a salt thereof, the method comprising: reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of the modified enzyme of claim 1.

5. The method according to claim 4, wherein the modified enzyme is a purified enzyme.

6. The method according to claim 4, wherein said reacting is performed using a transformed microorganism that produces the modified enzyme, or a treated product thereof.

7. The method according to claim 6, wherein the transformed microorganism comprises at least one of the following genetic modifications: (1) enhancement of ability to supply a fatty acid; (2) enhancement of ability to supply an amino acid; (3) enhancement of ability to supply ATP; and (4) deficiency or attenuation of an N-acylamino acid degrading enzyme.

8. The method according to claim 6, 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.

9. 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 bonded to each other by an amide bond, or a salt thereof, the method comprising culturing, in the presence of a carbon source, a microorganism comprising at least one of the following genetic modifications: (1) enhancement of ability to supply a fatty acid; (2) enhancement of ability to supply an amino acid; (3) enhancement of ability to supply ATP; and (4) deficiency or attenuation of an N-acylamino acid degrading enzyme, and wherein said microorganism comprises an expression unit comprising a polynucleotide encoding the modified enzyme of claim 1.

10. The method according to claim 9, wherein the enhancement of ability to supply a fatty acid is: (a) deficiency or attenuation of acyl CoA synthetase; and/or (b) enhancement of acyl-ACP thioesterase.

11. The method according to claim 10, wherein the acyl-ACP thioesterase has a thioesterase activity to lauroyl-ACP.

12. The method according to claim 10, wherein the acyl-ACP thioesterase is selected from: (i) a protein comprising: (i-1) the amino acid sequence of SEQ ID NO: 3 or (i-2) the amino acid sequence consisting of amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3; (ii) a protein comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acids in (ii-1) the amino acid sequence of SEQ ID NO: 3 or (ii-2) the amino acid sequence consisting of amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having an acyl-ACP thioesterase activity; and (iii) a protein comprising an amino acid sequence having 90% or more identity to (iii-1) the amino acid sequence of SEQ ID NO: 3 or (iii-2) the amino acid sequence consisting of amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having acyl-ACP thioesterase activity.

13. The method according to claim 9, wherein the carbon source is a saccharide.

14. The method according to claim 13, wherein the saccharide is glucose.

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

16. The method according to claim 15, wherein the bacterium belonging to the family Enterobacteriaceae is a bacterium belonging to the genus Escherichia or a bacterium belonging to the genus Pantoea.

17. The method according to claim 16, wherein the bacterium belonging to the genus Escherichia is Escherichia coli, and the bacterium belonging to the genus Pantoea is Pantoea ananatis.

18. The method according to claim 4, wherein the carboxyl group-containing compound is a fatty acid.

19. The method according to claim 18, wherein the fatty acid is a fatty acid having 8 to 18 carbon atoms.

20. The method according to claim 18, wherein the fatty acid is a fatty acid having 12 carbon atoms.

21. The method according to claim 18, wherein the fatty acid is a saturated fatty acid.

22. The method according to claim 4, wherein the amino group-containing compound is an amino acid, and the enzyme has an N-acylation activity to the amino acid.

23. The method according to claim 22, wherein the amino acid is L-glutamic acid or L-aspartic acid.

24. The method according to claim 4, wherein the carboxyl group-containing compound is lauric acid, and the N-acyl-amino group-containing compound is No-lauroyl-L-glutamic acid or No-lauroyl-L-aspartic acid.

25. The modified enzyme according to claim 1, wherein the mutation is selected from the group consisting of N101S, 1123T, Y134F, L137I, S161P, V174A, Q200E, V231A, V311A, M337G, A339G, S340A, Y344V, K388N, C576A, and combinations thereof.

26. The modified enzyme according to claim 25, further comprising an additional mutation selected from the group consisting of R117P, T122S, Y134V, V140I, C335S, T336S, M337A, Y344A, Y344G, Y344I, R350T, G379D, L390P, S455T, E483D, Q533R, and combinations thereof.

27. The modified enzyme according to claim 1, wherein the amino acid sequence of (B) comprises substitution, deletion, insertion, or addition of one to twenty amino acids, and the amino acid of (C) comprises an amino acid sequence having 97% or more identity to the amino acid sequence shown in SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram illustrating a structure of a plasmid pMW118-Sce-Km.

(2) FIG. 2 is a diagram illustrating a structure of a plasmid pMW118-Ptac-UcTEopt.

(3) FIG. 3 is a diagram illustrating a structure of a plasmid pMW118-PlacUV5-lacI-UcTEopt.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

(4) Modified Enzyme

(5) Described herein is a modified enzyme having an N-acylation activity.

(6) The modified enzyme is modified based on indole-3-acetic acid-amido synthetase GH3.6 (Q9LSQ4, hereinafter referred to as AtGH3-6) derived from Arabidopsis thaliana. AtGH3-6 was found as an enzyme that bonds a carboxyl group of indole-3-acetic acid and an amino group of a specific amino acid in an ATP-dependent manner to form an amide bond (Plant Cell 17:616-627 (2005)), and was also found to have an N-acylation activity including an ability to form an amide bond between a fatty acid and an amino acid. The modified enzyme has an N-acylation activity, the modified enzyme being obtained by modifying AtGH3-6 to improve a property related to the ability to produce an N-acyl-amino group-containing compound.

(7) The modified enzyme has: (A) a modified amino acid sequence that includes one or more mutations of predetermined amino acid residues in the amino acid sequence of SEQ ID NO: 1 (wild type AtGH3-6); (B) an amino acid sequence that includes substitution, deletion, insertion, or addition of one or several additional amino acid residues in the modified amino acid sequence described above; or (C) an amino acid sequence that includes additional mutations of one or more amino acid residues in the modified amino acid sequence as described above, and having 90% or more identity to the modified amino acid sequence, and the modified enzyme has an N-acylation activity as follows: (i) an N-acylation activity to L-glutamic acid and/or L-aspartic acid, and/or a substrate specificity to L-glutamic acid, wherein the activity is improved over an enzyme having the amino acid sequence of SEQ ID NO: 1.

(8) In another embodiment, the modified enzyme includes mutations of predetermined amino acid residues in any of (A) the amino acid sequence shown in SEQ ID NO: 1; (B) an amino acid sequence that includes substitution, deletion, insertion, or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, and (C) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, and the modified having an N-acylation activity as follows: an N-acylation activity to L-glutamic acid and/or L-aspartic acid, and/or (ii) a substrate specificity to L-glutamic acid, wherein the activity is improved over an enzyme having the amino acid sequence of SEQ ID NO: 1.

(9) In each of the amino acid sequences (B) and (B), one or several amino acid residues can be modified by one, two, three, or four kinds of mutations such as deletion, substitution, addition, and insertion of amino acid residues. The mutations of the amino acid residues may be introduced into one region or a plurality of different regions in the amino acid sequence. The term one or several amino acid residues refers to the number of the amino acid residues which do not largely impair an activity of the modified enzyme, such as, for example, an N-acylation activity. The term one or several refers to the number of, for example, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 (e.g., 1, 2, 3, 4, or 5).

(10) The identity percentage in each of the amino acid sequences (C) and (C) is 90% or more. The identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Calculation of the identity percentage of polypeptides (proteins) can be performed by Algorithm blastp. More specifically, calculation percentage of the identity of polypeptides can be performed using Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11 Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) as default settings in Algorithm blastp provided in National Center for Biotechnology Information (NCBI). Calculation of the identity percentage of polynucleotides (genes) can be performed by Algorithm blastn. More specifically, calculation of the identity percentage of polynucleotides can be performed using Scoring Parameters (Match/Mismatch Scores=1,2; Gap Costs=Linear) as default settings in Algorithm blastn provided in NCBI.

(11) The N-acylation activity (also referred to as N-acylase activity) refers to an activity of bonding an amino group-containing compound and a carboxyl group-containing compound as substrates to each other by an amide bond to produce an N-acyl-amino group-containing compound. The N-acylation activity may be, for example, an activity of producing an N-acylamino acid from an amino acid, e.g. an -L-amino acid described below, and a fatty acid, e.g. a saturated fatty acid described below, and an activity of producing N-lauroyl-L-glutamic acid or N-lauroyl-L-aspartic acid from L-glutamic acid or L-aspartic acid, and lauric acid may be used as an index. The phrase having an N-acylation activity may mean having an activity of, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 94% or more, 96% or more, 98% or more, or an activity which is equivalent (that is, 100%) or more based on an activity of an enzyme having the amino acid sequence of SEQ ID NO: 1 when the activity is measured under specific measurement conditions. As such specific measurement conditions, the following conditions can be adopted. An enzyme to be measured is prepared as a purified enzyme. A 0.2 mL reaction solution having a pH of 8.0, the 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 MgCl.sub.2, 1 mM DTT, and 50 g/mL purified enzyme, is incubated at 25 C. for 24 hours. After completion of the reaction, 0.8 mL of a reaction stop solution (1.4% (w/v) phosphoric acid, 75% (v/v) methanol) is added to the reaction solution. The supernatant after centrifugation is subjected to UPLC-MS analysis, and a molecular weight signal that coincides with that of an N-acylamino acid, e.g. N-lauroyl-L-glutamic acid or N-lauroyl-L-aspartic acid, is measured to evaluate an N-acylation activity.

(12) In each of the amino acid sequences (B), (B), (C), and (C), a mutation may be introduced into a site in a catalytic domain and into a site other than the catalytic domain as long as the modified enzyme can maintain the target property. The position of an amino acid residue into which a mutation may be introduced and at which the modified enzyme can maintain the target property is obvious to a person skilled in the art. Specifically, a person skilled in the art can 1) compare amino acid sequences of a plurality of proteins having homogeneous property with each other, 2) clarify a relatively conserved region and a relatively non-conserved region, and then 3) predict a region capable of playing an important role for a function and a region incapable of playing an important role for a function from the relatively conserved region and the relatively non-conserved region, respectively, and can thus recognize structure-function correlation. Therefore, a person skilled in the art can identify the position of an amino acid residue into which a mutation may be introduced in the amino acid sequence of the protein used.

(13) When an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be conservative substitution. The term conservative substitution when used in the present specification refers to replacing a certain amino acid residue with an amino acid residue having a similar side chain. Families of the amino acid residue having a similar side chain are known in the field concerned. Examples of such families include an amino acid having a basic side chain, e.g. lysine, arginine, or histidine, an amino acid having an acidic side chain, e.g. aspartic acid or glutamic acid, an amino acid having an uncharged polar side chain, e.g. asparagine, glutamine, serine, threonine, tyrosine, or cysteine, an amino acid having a nonpolar side chain, e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan, an amino acid having a -position-branched side chain, e.g. threonine, valine, or isoleucine, an amino acid having an aromatic side chain, e.g. tyrosine, phenylalanine, tryptophan, or histidine, an amino acid having a hydroxy group, e.g. alcoholic- or phenolic-containing side chain, e.g. serine, threonine, or tyrosine, and an amino acid having a sulfur-containing side chain, e.g. cysteine or methionine. The conservative substitution of an amino acid may be a substitution between aspartic acid and glutamic acid, substitution among arginine, lysine, and histidine, substitution between tryptophan and phenylalanine, substitution between phenylalanine and valine, substitution among leucine, isoleucine, and alanine, or substitution between glycine and alanine.

(14) The modified enzyme may also be a fusion protein linked to a heterologous moiety via a peptide bond. Examples of such a heterologous moiety include a peptide component that facilitates purification of a target protein, e.g. a tag portion such as a histidine tag or Strep tag II; or a protein used for purification of a target protein, such as glutathione-S-transferase, a maltose binding protein, or mutants thereof, a peptide component that improves a solubility of a target protein, e.g. Nus-tag, a peptide component that acts as a chaperon, e.g. a trigger factor, a peptide component recognized by a protease for cleaving the purification tag, e.g. a Thrombin recognition sequence or a TEV protease recognition sequence, a peptide component having other functions, e.g. a full-length protein or a part thereof, and a linker.

(15) The mutation of a predetermined amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 or any of the amino acid sequences (A), (B), and (C) is a mutation that improves a property related to an ability to produce an N-acyl-amino group-containing compound in the modified enzyme. Examples of the mutation of a predetermined amino acid residue include mutations of amino acid residues N101, R117, T122, 1123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specific examples of the mutation of a predetermined amino acid residue include substitutions of 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 mutations of predetermined amino acid residues contained in the modified enzyme is 1 or more, and may be, for example, 1 to 26, 1 to 20, 1 to 15, or 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

(16) Examples of the property related to the ability to produce an N-acyl-amino group-containing compound, the property being improved by mutation of a predetermined amino acid residue, include an N-acylation activity to a specific amino acid substrate and a substrate specificity to a specific amino acid substrate. The degree of improvement is indicated by comparison with an enzyme having the amino acid sequence of SEQ ID NO: 1, and may be measured as comparison between fusion proteins linked to a heterologous moiety via a peptide bond.

(17) Examples of the N-acylation activity to a specific amino acid substrate include an N-acylation activity to L-glutamic acid and an N-acylation activity to L-aspartic acid. A carboxyl group-containing compound that is a substrate used for measuring the N-acylation activity may be, for example, a fatty acid, and is a saturated fatty acid, lauric acid. The N-acylation activity can be measured, for example, under the measurement conditions described above. The degree of improvement in the N-acylation activity to a specific amino acid substrate is not particularly limited as long as the N-acylation activity exceeds an N-acylation activity of an enzyme having the amino acid sequence of SEQ ID NO: 1, but is, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, and at most 2 times or more.

(18) Examples of the mutation that improves the N-acylation activity to L-glutamic acid or L-aspartic acid include mutations of amino acid residues N101, R117, T122, 1123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specific examples of the mutation that improves the N-acylation activity to L-glutamic acid or L-aspartic acid include substitutions of 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.

(19) Examples of the substrate specificity to a specific amino acid substrate include a substrate specificity to L-glutamic acid. The substrate specificity to L-glutamic acid can be indicated as an activity ratio between an N-acylation activity to L-glutamic acid and an N-acylation activity to 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, and is a saturated fatty acid, lauric acid. The N-acylation activity can be measured, for example, under the measurement conditions described above. The degree of improvement in the substrate specificity to a specific amino acid substrate is not particularly limited as long as the substrate specificity exceeds a substrate specificity of an enzyme having the amino acid sequence of SEQ ID NO: 1, but is, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, or 2 times or more.

(20) Examples of the mutation that improves the substrate specificity to L-glutamic acid include mutations of amino acid residues N101, R117, T122, 1123, Y134, L137, V140, S161, V174, Q200, V231, V311, C335, T336, M337, A339, S340, Y344, R350, G379, K388, L390, S455, E483, Q533, and C576. More specific examples of the mutation that improves the substrate specificity to L-glutamic acid include substitutions of 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.

(21) Polynucleotide

(22) The present invention also provides a polynucleotide encoding the modified enzyme as described herein. The polynucleotide may be DNA or RNA, but DNA is a particular example.

(23) The polynucleotide encoding the enzyme may be a polynucleotide such as: (a) a polynucleotide that includes the nucleotide sequence shown in SEQ ID NO: 2 (wild type AtGH3-6); (b) a polynucleotide that hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions; (c) a polynucleotide that includes a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 2; and (d) a degenerate variant of any of the polynucleotide described above,

(24) The polynucleotide may encode a modified enzyme, and the polynucleotide may include mutations of nucleotide sequences corresponding to mutations of the predetermined amino acid residues described above, and wherein the modified enzyme has an N-acylation activity as follows: (i) an N-acylation activity to L-glutamic acid and/or L-aspartic acid; and/or (ii) a substrate specificity to L-glutamic acid, wherein the activity is improved over an enzyme having the amino acid sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 2 encodes the amino acid sequence of SEQ ID NO: 1.

(25) In the polynucleotide (b), the term stringent conditions refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Examples of the stringent conditions include hybridization at about 45 C. in 6SSC (sodium chloride/sodium citrate) followed by washing once or more at 50 to 65 C. in 0.2 SSC and 0.1% SDS.

(26) In the polynucleotide (c), the identity percentage of a nucleotide sequence to the nucleotide 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.

(27) In the polynucleotide (d), the term degenerate variant refers to a polynucleotide mutant in which at least one codon encoding a certain amino acid residue in a polynucleotide before mutation has been changed into another codon encoding the same amino acid residue. Since such a degenerate variant is a variant based on a silent mutation, a protein (enzyme) encoded by the degenerate variant is the same as a protein (enzyme) encoded by a polynucleotide before mutation.

(28) The degenerate variant is a polynucleotide mutant in which a codon has been changed so as to be adapted to a codon usage frequency of a host cell into which the degenerate variant is to be introduced. When a gene is expressed by a heterologous host cell, e.g. a microorganism, due to a difference in codon usage frequency, a corresponding tRNA molecular species is not sufficiently supplied, which may cause a reduction in translation efficiency and/or incorrect translation, e.g. stop of translation. In Escherichia coli, for example, low frequency codons listed in Table 1 are known.

(29) TABLE-US-00001 TABLE1 LowfrequencycodoninEscherichiacoli Aminoacid Low residue Codon frequencycodon Arg AGG/AGA/CGG/CGA/CGU/CGC AGG/AGA/CGG/CGA Gly GGG/GGA/GGU/GGC GGA Ile AUA/AUU/AUC AUA Leu UUG/UUA/CUG/CUA/CUU/CUC CUA Pro CCG/CCA/CCU/CCC CCC

(30) Therefore, the present invention can use a degenerate variant adapted to the codon usage frequency of a host cell described below. In the degenerate variant, for example, a codon or codons encoding one or more kinds of amino acid residues such as an arginine residue, a glycine residue, an isoleucine residue, a leucine residue, and a proline residue may be changed. More specifically, in the degenerate variant, one or more kinds of low frequency codons, e.g. AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC, may be changed. The degenerate variant may contain changes of one or more kinds, e.g. one, two, three, four, or five kinds, of codons such as: i) a change of at least one of four kinds of codons, AGG, AGA, CGG, and CGA encoding Arg into another codon, CGU or CGC encoding Arg; ii) a change of one kind of codon, GGA encoding Gly into another codon, GGG, GGU, or GGC; iii) a change of one kind of codon, AUA encoding Ile into another codon, AUU or AUC; iv) a change of one kind of codon, CUA encoding Leu into another codon, UUG, UUA, CUG, CUU, or CUC; and v) a change of one kind of codon, CCC encoding Pro into another codon, CCG, CCA, or CCU.

(31) When the degenerate variant is RNA, the nucleotide residue U should be used as described above, whereas when the degenerate variant is DNA, T should be used in place of the nucleotide residue U. The number of mutations of nucleotide residues for being adapted to the codon usage frequency of a host cell, which is not particularly limited as long as the same protein is encoded before and after mutation, is, for example, 1 to 400, 1 to 300, 1 to 200, or 1 to 100.

(32) A low frequency codon can be easily identified based on the kind and genome sequence information of any host cell by using techniques known in the field concerned. Therefore, the degenerate variant may contain a change of a low frequency codon into a non-low frequency codon, e.g. a high frequency codon. In addition, since a method for designing a mutant in consideration of a factor such as adaptability to the genomic GC content of a producing strain as well as a low frequency codon is known (Alan Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments, BMC Bioinformatics. 2006 Jun. 6; 7:285), such a method may be used. Thus, the mutant described above can be appropriately prepared in accordance with any kind of host cell, e.g. a microorganism described below, into which the mutant can be introduced.

(33) Expression Vector

(34) Described herein is an expression vector. The expression vector contains the polynucleotide as described herein or a polynucleotide encoding the modified enzyme.

(35) The term expression unit refers to a minimum unit that contains a certain polynucleotide to be expressed as a protein, and a promoter operably linked thereto, and enables transcription of the polynucleotide and consequently production of a protein encoded by the polynucleotide. The expression unit may further contain an element such as a terminator, a ribosome binding site, or a drug-resistant gene. The expression unit may be DNA or RNA, and DNA is a particular example. The expression unit may be homologous, that is, inherent, or heterologous, that is, non-inherent, relative to a host cell. The expression unit may be an expression unit containing one polynucleotide to be expressed as a protein and a promoter operably linked thereto, that is, an expression unit that enables expression of monocistronic mRNA, or an expression unit containing a plurality of polynucleotides to be expressed as a protein, e.g. 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more polynucleotides, and a promoter operably linked thereto, that is, an expression unit that enables expression of polycistronic mRNA. The expression unit can be contained in a genomic region, e.g., a natural genomic region that is a natural locus in which a polynucleotide encoding the protein is inherently present, or a non-natural genomic region that is not the natural locus, or a non-genomic region, e.g. in a cytoplasm, in a microorganism or host cell. The expression unit may be contained in a genomic region at one or more, e.g. 1, 2, 3, 4, or 5, different positions. Examples of a specific form of the expression unit contained in the non-genomic region include a plasmid, a viral vector, a phage, and an artificial chromosome.

(36) The promoter present in the expression unit is not particularly limited as long as the promoter can express a protein (enzyme) encoded by a polynucleotide linked to a downstream side thereof in a host cell. For example, the promoter may be homologous or heterologous relative to a host cell. For example, a constitutive or inducible promoter generally used for production of a recombinant protein can be used. Examples of such a promoter include a PhoA promoter, a PhoC promoter, a T7 promoter, a T5 promoter, a T3 promoter, a lac promoter, a trp promoter, a tre promoter, a tac promoter, a PR promoter, a PL promoter, an SP6 promoter, an arabinose-inducible promoter, a cold shock promoter, and a tetracycline-inducible promoter. A promoter having a strong transcription activity in a host cell can be used. Examples of the promoter having a strong transcription activity in a host cell include a promoter of a gene highly expressed in the host cell and a promoter derived from a virus.

(37) The expression vector may further contain, as the expression unit, an element such as a terminator that functions in a host cell, a ribosome binding site, or a drug-resistant gene in addition to the minimum unit described above. Examples of the drug-resistant gene include a gene resistant against a drug such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, or chloramphenicol.

(38) The expression vector may further contain a region which enables homologous recombination with a genome of a host cell for homologous recombination with the genome DNA of the host cell. For example, the expression vector may be designed such that an expression unit contained therein is positioned between a pair of homologous regions, e.g., homology arms homologous to a specific sequence in a genome of a host cell, loxP or FRT. The genome region, such as a target of the homologous region, of the host cell into which the expression unit is to be introduced is not particularly limited, but may be a locus of a gene having a large amount of expression in the host cell.

(39) The expression vector may be a plasmid, a virus vector, a phage, or an artificial chromosome. The expression vector may be an integrative vector or a non-integrative vector. The integrative vector may be a vector the entire of which is incorporated into a genome of a host cell. Alternatively, the integrative vector may be a vector only a part of which is incorporated into a genome of a host cell, such as an expression unit. Furthermore, the expression vector may be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may be a generally used expression vector. Examples of such an expression vector include pUC, e.g. pUC19 or pUC18, pSTV, pBR, e.g. pBR322, pHSG, e.g., pHSG299, pHSG298, pHSG399, or pHSG398, RSF, e.g. RSF1010, pACYC, e.g. pACYC177 or pACYC184, pMW, e.g. pMW119, pMW118, pMW219, or pMW218, pQE, e.g. pQE30, pET, e.g. pET28a, and derivatives thereof.

(40) Host Cell

(41) Described herein is a host cell. The host cell contains an expression unit of a polynucleotide encoding the modified enzyme. The host cell contains an expression unit containing a polynucleotide encoding the modified enzyme and a promoter operably linked thereto. The host cell is a microorganism.

(42) The host cell is a transformed microorganism. Examples of the host cell include a bacterium such as a bacterium belonging to the family Enterobacteriaceae, and fungi. The bacterium may also be a Gram-positive bacterium or a Gram-negative bacterium. Examples of the gram-positive bacterium include a bacterium belonging to the genus Bacillus and a bacterium belonging to the genus Corynebacterium. The bacterium belonging to the genus Bacillus can be Bacillus subtilis. The bacterium belonging to the genus Corynebacterium can be Corynebacterium glutamicum. Examples of the Gram-negative bacterium include a bacterium belonging to the genus Escherichia and a bacterium belonging to the genus Pantoea. The bacterium belonging to the genus Escherichia can be Escherichia coli. The bacterium belonging to the genus Pantoea can be Pantoea ananatis. The fungus can be a microorganism belonging to the genus Saccharomyces or belonging to the genus Schizosaccharomyces. The microorganism belonging to the genus Saccharomyces can be Saccharomyces cerevisiae. The microorganism belonging to the genus Schizosaccharomyces can be Schizosaccharomyces pombe. The host cell is a bacterium belonging to the family Enterobacteriaceae, a bacterium belonging to the genus Escherichia or belonging to the genus Pantoea, and Escherichia coli or Pantoea ananatis.

(43) In one embodiment, the host cell can be used for producing an N-acyl-amino group-containing compound or a salt thereof by the modified enzyme produced in the host cell using the host cell itself, e.g. a cultured product of the host cell, or a treated product thereof, e.g. a disrupted product, a lysate, or a lyophilizate of the host cell. In another embodiment, the host cell can be used for obtaining the modified enzyme as an unpurified, crude, or purified enzyme.

(44) When the host cell is used for producing an N-acyl-amino group-containing compound or a salt thereof, the host cell may be, for example, a host having enhanced ability to incorporate an amino acid and/or a fatty acid in order to improve supply efficiency of a substrate of an enzymatic reaction to improve production efficiency. Examples of the host having the enhanced incorporating ability include a host that produces or enhances a protein such as an enzyme related to the incorporating ability. Examples of the host that produces or enhances a protein such as an enzyme related to the incorporating ability include a host into which an expression unit of the protein is introduced by transformation, a host containing a mutation that enhances the amount of expression of the protein in a host genome, and a host containing a mutation that enhances an activity of the protein in the host genome. Such a host cell can be used for producing 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 solution 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 a host cell can be used for producing (direct fermentation method) 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 solution containing a carbon source, e.g. a saccharide such as glucose.

(45) In order to suppress a loss of a substrate of an enzymatic reaction and/or to promote supply of the substrate of the enzymatic reaction or to suppress a loss of a product, the host cell may be, for example, a host cell having at least one genetic modification such as: (1) enhancement of ability to supply a fatty acid; (2) enhancement of ability to supply an amino acid; (3) enhancement of ability to supply ATP; and (4) deficiency or attenuation of an N-acylamino acid degrading enzyme.

(46) The host cell having a genetic modification effective for enhancement of ability to supply a fatty acid may be a host cell in which a fatty acid degradation system is attenuated or deficient and/or a fatty acid synthesis system is enhanced.

(47) Examples of the host cell in which the fatty acid degradation system is attenuated or deficient include a host cell in which a protein such as an enzyme related to the degradation system is attenuated or deficient and a host cell that produces an inhibitor of a protein such as an enzyme related to the degradation system.

(48) Examples of the host cell in which a protein such as an enzyme related to the fatty acid degradation system is attenuated or deficient include a host cell containing a mutation that reduces or deletes the amount of expression of the protein in a host cell genome and a host cell containing a mutation that reduces or deletes an activity of the protein in the host cell genome.

(49) Examples of the host cell that produces or enhances an inhibitor of a protein such as an enzyme related to the fatty acid degradation system include a host cell into which an expression unit of the inhibitor is introduced by transformation, a host cell containing a mutation that enhances the amount of expression of the inhibitor in a host cell genome, and a host cell containing a mutation that enhances an activity of the inhibitor in the host cell genome.

(50) More specifically, examples of the protein such as an enzyme related to the fatty acid degradation system include acyl CoA synthetase (fadD), acyl CoA dehydrogenase (fadE), enoyl CoA hydratase (fadB and fadJ), 3-hydroxyacyl CoA dehydrogenase (fadB and fadJ), and 3-ketoacyl CoA thiolase (fadA and fadI). Note that the name in the parenthesis after the enzyme name is a gene name thereof and the same applies to the following description. Among these enzymes, for example, acyl CoA synthetase is preferable.

(51) Examples of the host cell in which the fatty acid synthesis system is enhanced include a host cell that produces or enhances a protein such as an enzyme related to the synthesis system.

(52) Examples of the host cell that produces or enhances a protein such as an enzyme related to the synthesis system include a host cell into which an expression unit of the protein is introduced by transformation, a host cell containing a mutation that enhances the amount of expression of the protein in a host cell genome, and a host cell containing a mutation that enhances an activity of the protein in the host cell genome.

(53) More specifically, examples of the protein such as an enzyme related to the fatty acid synthesis system include acyl-ACP thioesterase, acetyl COA carboxylase (accABCD), malonyl CoA-ACP transacylase (fabD), -ketoacyl ACP synthase III (fabH), -ketoacyl ACP reductase (fabG), -hydroxyacyl ACP dehydratase (fabZ), enoyl ACP reductase (fabI), -ketoacyl ACP synthase I (fabB), and -ketoacyl ACP synthase II (fabF). Among these enzymes, for example, acyl-ACP thioesterase is a particular example.

(54) The genetic modification for enhancement of ability to supply a fatty acid may be. (a) deficiency or attenuation of acyl CoA synthetase; and/or (b) enhancement of acyl-ACP thioesterase.

(55) Examples of the deficiency or attenuation of acyl CoA synthetase include deletion or mutagenesis of a fadD gene on a host cell genome.

(56) Examples of the enhancement of acyl-ACP thioesterase include introduction of an acyl-ACP thioesterase expression unit into a host cell.

(57) The acyl-ACP thioesterase may be, for example, an acyl-ACP thioesterase having a thioesterase activity to lauroyl-ACP. In addition, the acyl-ACP thioesterase may be, for example, a protein as follows: (i) a protein including (i-1) an amino acid sequence of SEQ ID NO: 3 or (i-2) the amino acid sequence having the amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3; (ii) a protein including an amino acid sequence that includes substitution, deletion, insertion, or addition of one or several amino acids in (ii-1) the amino acid sequence of SEQ ID NO: 3 or (ii-2) the amino acid sequence having amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having an acyl-ACP thioesterase activity; and (iii) a protein including an amino acid sequence having 90% or more identity to (iii-1) the amino acid sequence of SEQ ID NO: 3 or (iii-2) the amino acid sequence having amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3, and having acyl-ACP thioesterase activity.

(58) The host cell having a genetic modification effective for enhancement of ability to supply an amino acid may be a host cell in which an amino acid degradation system is attenuated or deficient and/or an amino acid synthesis system is enhanced.

(59) Examples of the host cell in which an amino acid degradation system is attenuated or deficient include a host cell in which a protein such as an enzyme related to the amino acid degradation system is attenuated or deficient and a host cell that produces an inhibitor of a protein such as an enzyme related to the amino acid degradation system.

(60) Examples of the host cell in which a protein such as an enzyme related to the amino acid degradation system is attenuated or deficient include a host cell containing a mutation that reduces or deletes the amount of expression of the protein in a host cell genome and a host cell containing a mutation that reduces or deletes an activity of the protein in the host cell genome.

(61) Examples of the host cell that produces or enhances an inhibitor of a protein such as an enzyme related to the amino acid degradation system include a host cell into which an expression unit of the inhibitor is introduced by transformation, a host cell containing a mutation that enhances the amount of expression of the inhibitor in a host cell genome, and a host cell containing a mutation that enhances an activity of the inhibitor in the host cell genome.

(62) More specifically, the protein such as an enzyme related to the amino acid degradation system may be an enzyme that catalyzes a reaction of branching from a biosynthetic pathway of a target amino acid, e.g. L-glutamic acid to produce a compound other than the target amino acid. Examples of such an enzyme include -ketoglutarate dehydrogenase (sucA), isocitrate lyase (aceA), succinate dehydrogenase (sdhABCD), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyate synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and 1-pyrroline-5-carboxylate dehydrogenase (putA). Among these enzymes, for example, -ketoglutarate dehydrogenase is a particular example.

(63) Examples of the host cell in which the amino acid synthesis system is enhanced include a host cell that produces or enhances a protein such as an enzyme related to the amino acid synthesis system.

(64) Examples of the host cell that produces or enhances a protein such as an enzyme related to the amino acid synthesis system include a host cell into which an expression unit of a protein of the amino acid is introduced by transformation, a host cell containing a mutation that enhances the amount of expression of the protein in a host cell genome, and a host cell containing a mutation that enhances an activity of the protein in the host cell genome.

(65) More specifically, examples of the protein such as an enzyme related to the amino acid, e.g. glutamic acid synthesis system include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA and acnB), citrate synthase (gltA), methyl citrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate kinase (pykA and pykF), pyruvate dehydrogenase (aceEF and lpdA), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA and pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosphosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA and pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase.

(66) The host cell having a genetic modification effective for enhancement of ability to supply ATP may be a host cell in which an ATP degradation system is attenuated or deficient and/or an ATP synthesis system is enhanced.

(67) Examples of the host cell in which the ATP degradation system is attenuated or deficient include a host cell in which a protein such as an enzyme related to the ATP degradation system is attenuated or deficient.

(68) Examples of the host cell in which a protein such as an enzyme related to the ATP degradation system is attenuated or deficient include a host cell containing a mutation that reduces or deletes the amount of expression of the protein in a host cell genome and a host cell containing a mutation that reduces or deletes an activity of the protein in the host cell genome.

(69) Examples of the host cell in which the ATP synthesis system is enhanced include a host cell that produces or enhances a protein such as an enzyme related to the synthesis system.

(70) Examples of the host cell that produces or enhances a protein such as an enzyme related to the ATP synthesis system include a host cell into which an expression unit of the protein is introduced by transformation, a host cell containing a mutation that enhances the amount of expression of the protein in a host cell genome, and a host cell containing a mutation that enhances an activity of the protein in the host cell genome.

(71) Examples of a genetic modification effective for deficiency or attenuation of an N-acylamino acid degrading enzyme include deficiency or attenuation of acylase. Examples of the deficiency or attenuation of acylase include deletion or mutagenesis of an acylase gene on a host cell genome.

(72) Such a host cell can be used for producing, such as by e.g. an enzyme method, an N-acyl-amino group-containing compound, e.g. an N-acylamino acid, or a salt thereof in a reaction solution containing an amino group-containing compound, e.g. an amino acid, and a carboxyl group-containing compound, e.g. a fatty acid, as a transformed microorganism, e.g. a cultured product of the microorganism, that produces the enzyme or a treated product thereof, e.g. a disrupted product, a lysate, or a lyophilizate of the microorganism. Such a host cell can be used for producing 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 solution 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 a host cell has an enhanced metabolic pathway for producing an amino group-containing compound, e.g. an amino acid, and/or a carboxyl group-containing compound, e.g. a fatty acid, from a carbon source, e.g. a saccharide such as glucose, and therefore can be used for producing, such as by a direct fermentation method, 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 solution containing a carbon source.

(73) The transformed microorganism can be produced by any method known in the field concerned. For example, such a transformed microorganism as described above can be produced by a method using an expression vector, such as by, e.g. a competent cell method or an electroporation method, or a genome modification technique. When the expression vector is an integrative vector that causes homologous recombination with the genome DNA of a host cell, an expression unit can be incorporated into the genome DNA of the host cell by transformation. On the other hand, when the expression vector is a non-integrative vector that does not cause homologous recombination with the genome DNA of the host cell, the expression unit is not incorporated into the genome DNA of the host cell by transformation, and can exist independently of the genome DNA in the host cell in the state of the expression vector. Alternatively, according to genome editing technology, such as e.g. CRISPR/Cas system, Transcription Activator-Like Effector Nucleases (TALEN), it is possible to incorporate an expression unit into the genome DNA of a host cell and to modify an expression unit inherently contained in the host cell.

(74) Method for Producing N-Acyl-Amino Group-Containing Compound or Salt Thereof

(75) Described herein is a method for producing an N-acyl-amino group-containing compound or a salt thereof. The method includes producing an N-acyl-amino group-containing compound or a salt thereof by reacting an amino group-containing compound and a carboxyl group-containing compound in the presence of a modified enzyme having an N-acylation activity.

(76) The modified enzyme used in the method may be the modified enzyme described above.

(77) Amino Group-Containing Compound

(78) The amino group-containing compound that can be used in the method may be either an organic compound containing an amino group in which a nitrogen atom is bonded to one or two hydrogen atoms, or an organic compound containing an amino group in which a nitrogen atom is not bonded to a hydrogen atom. The amino group-containing compound is a compound containing an amino group in which a nitrogen atom is bonded to one or two hydrogen atoms, andcan be a compound containing an amino group in which a nitrogen atom is bonded to two hydrogen atoms from a viewpoint of a substrate specificity of the enzyme, and the like.

(79) The amino group-containing compound that can be used in the method can be an amino group-containing compound having an anionic group. Examples of the anionic group include a carboxyl group, a sulfonic acid group, a sulfuric acid group, and a phosphoric acid group.

(80) Examples of the amino group-containing compound having a carboxyl group as an anionic group include an amino acid and a peptide.

(81) Examples of the amino acid include an -amino acid, a -amino acid, and a Y-amino acid. Examples of the x-amino acid 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 the -amino acid include -alanine. Examples of the -amino acid include -aminobutyric acid. The amino group of the amino acid may be any of an amino group in which a nitrogen atom is bonded to two hydrogen atoms, an amino group in which a nitrogen atom is bonded to one hydrogen atom, and an amino group in which a nitrogen atom is not bonded to a hydrogen atom. Examples of the amino acid containing an amino group in which a nitrogen atom is bonded to one hydrogen atom include sarcosine, N-methyl--alanine, N-methyltaurine, and proline. The amino acid may be either an L-amino acid or a D-amino acid.

(82) The peptide is a compound having a structure in which the above-described amino acids are linked to each other by an amide bond. Examples of the peptide include an oligopeptide, e.g. dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, or octapeptide, having a structure in which 2 to 10 amino acids are linked to each other by an amide bond, and a polypeptide (protein) having a structure in which 11 or more amino acids are linked to each other by an amide bond. Examples of the dipeptide include aspartylphenylalanine, glycylglycine, -alanylhistidine, and alanylglutamine.

(83) Examples of the amino group-containing compound having a sulfonic acid group as an anionic group include taurine, N-methyl taurine, and cysteic acid.

(84) Examples of the amino group-containing compound having a sulfuric acid group as an anionic group include O-sulfoserine and O-sulfothreonine.

(85) Examples of the amino group-containing compound having a phosphoric acid group as an anionic group include ethanolamine phosphate, phosphoserine, and phosphothreonine.

(86) The amino group-containing compound that can be used in the method can be an acidic amino acid. The acidic amino acid refers to an amino acid having an acidic side chain. Examples of the acidic amino acid include glutamic acid and aspartic acid, and glutamic acid is preferable.

(87) Carboxyl Group-Containing Compound

(88) The carboxyl group-containing compound that can be used in the method is a compound containing a carboxyl group without a substituent, such as e.g. a free form, an ion, or a salt. Examples of the carboxyl group-containing compound include a fatty acid, an aromatic carboxylic acid, an indole carboxylic acid, and a mixture thereof. The carboxyl group-containing compound can be a fatty acid.

(89) The fatty acid may be, for example, a fatty acid having 8 to 18 carbon atoms, and can be a fatty acid having 12 carbon atoms. Examples of the fatty acid 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), hexadecenoic acid (C16: 1) (e.g., palmitoleic acid or sapienic acid), margaric acid (C17), stearic acid (C18), octadecenoic acid (C18:1) (e.g., oleic acid or vaccenic acid), linoleic acid (C18: 2), and linolenic acid (C18: 3), e.g. -linolenic acid or -linolenic acid. The number in parentheses indicates the number of carbon atoms. In addition to these, a mixed fatty acid such as a coconut oil fatty acid, a palm fatty acid, or a hydrogenated beef tallow fatty acid can also be used.

(90) The fatty acid may be a saturated fatty acid. Among the 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. The saturated fatty acid can be lauric acid. The fatty acid may be an unsaturated saturated fatty acid. Among the fatty acids, examples of the unsaturated fatty acid 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 or sapienic acid, octadecenoic acid (C18: 1), e.g. oleic acid or vaccenic acid, linoleic acid (C18: 2), and linolenic acid (C18: 3), e.g. -linolenic acid or -linolenic acid. The number in parentheses indicates the number of carbon atoms. The unsaturated fatty acid can be dodecenoic acid (C12: 1) or hexadecenoic acid (C16: 1). A mixture of these fatty acids may be used as the fatty acid.

(91) Examples of the aromatic carboxylic acid include benzoic acid, phthalic acid, isophthalic acid, terephthalic acid, salicylic acid, gallic acid, cinnamic acid, and a mixture thereof.

(92) N-Acyl-Amino Group-Containing Compound or Salt Thereof

(93) The N-acyl-amino group-containing compound or a salt thereof produced by the method is a compound having a structure in which an amino group of the amino group-containing compound and a carboxyl group of the carboxyl group-containing compound form an amide bond. The N-acyl-amino group-containing compound or a salt thereof is produced by a reaction between the amino group-containing compound and the carboxyl group-containing compound in the presence of the enzyme. The amino group that reacts with a carboxyl group may be at any position of the amino group-containing compound, and may be, for example, at any of an -position, a -position, a -position, a -position, and an -position. The N-acyl-amino group-containing compound or a salt thereof produced by the method can be N-acyl-L-glutamic acid or N-acyl-L-aspartic acid, or a salt thereof, or N-lauroyl-L-glutamic acid or N-lauroyl-L-aspartic acid, or a salt thereof. Examples of the salt of the N-acyl-amino group-containing compound include an inorganic salt and an organic salt. Examples of the inorganic salt include a salt of a metal, e.g., a monovalent metal such as lithium, sodium, potassium, rubidium, or cesium, or a divalent metal such as calcium, magnesium, or zinc, and a salt of an inorganic base, e.g. ammonia. Examples of the organic salt include a salt of an organic base, e.g. ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, triethanolamine, lysine, arginine, histidine, or ornithine.

(94) The acyl having n carbon atoms refers to an acyl represented by C.sub.n-1H.sub.mCO, in which a hydrogen atom may be replaced. Here, m is appropriately determined according to the number of carbon atoms and the presence or absence of an unsaturated bond.

(95) The N-monounsaturated acyl-amino group-containing compound, wherein the unsaturated acyl is a monounsaturated acyl having 10 to 16 carbon atoms refers to a compound represented by C.sub.n-1H.sub.mCONHCHRCOOH, in which the hydrogen atom represented by H.sub.m may be replaced, m is as defined above, and R represents a side chain of an acidic amino acid, as a free form. That is, the N-monounsaturated acyl-amino group-containing compound refers to a compound in which one hydrogen atom on an amino group of an acidic amino acid is replaced 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 the N-monounsaturated acyl-amino group-containing compound.

(96) The N-monounsaturated acyl-amino group-containing compound may be derived from a fatty acid represented by C.sub.n-1H.sub.mCOOH or a derivative thereof for convenience. In addition, in the present specification, an unsaturated acyl having n carbon atoms and a fatty acid and the like from which the unsaturated acyl having n carbon atoms is derived may be represented by Cn: m. For example, N-dodecenonyl acidic amino acid (C12: 1) refers to a compound in which one hydrogen atom on an amino group of an acidic amino acid is replaced with a dodecenonyl group which is an acyl group derived from dodecenoic acid (C12: 1), C.sub.11H.sub.21CONHCHRCOOH in which R represents a side chain of the acidic amino acid.

(97) When the N-acyl-amino group-containing compound or a salt thereof produced by the method 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). N-dodecenoyl acidic amino acid (C12: 1) or N-hexadecenoyl acidic amino acid (C16: 1) is preferable. N-dodecenoyl glutamic acid (C12: 1) or N-hexadecenoyl glutamic acid (C16: 1) are particular examples.

(98) The N-monounsaturated acyl-amino group-containing compound may include one kind of N-monounsaturated acyl-amino group-containing compound, or two or more kinds of N-monounsaturated acyl-amino group-containing compounds. That is, the N-monounsaturated acyl-amino group-containing compound may include one or more 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 kinds of N-monounsaturated acyl-amino group-containing compounds are used, a combination of N-dodecenoyl acidic amino acid (C12: 1) and one or more of N-decenoyl acidic amino acid (C10: 1), N-tetradecenoyl acidic amino acid (C14: 1), and N-hexadecenoyl acidic amino acid (C16: 1) is a particular example, and a combination of N-dodecenoyl acidic amino acid (C12: 1) and N-hexadecenoyl acidic amino acid (C16: 1) is a particular example from the viewpoint of improving foam quality.

(99) The N-monounsaturated acyl-amino group-containing compound is a compound represented by the following general formula (A):

(100) ##STR00001##

(101) In general formula (A), 1 is an integer of 1 or 2, and m is an integer of 0 to 6.

(102) The stereochemistry (unsaturated double bond site) of the N-monounsaturated acyl-amino group-containing compound may be either in a cis or trans conformation. However, cis is a particular example from the viewpoint of suppressing an increase in viscosity and achieving a low viscosity with high handleability.

(103) Production of N-Acyl-Amino Group-Containing Compound by Enzyme Method

(104) As the enzyme used in the method, a recombinant protein can be used. The recombinant protein can be obtained, for example, using a cell-free vector or from a microorganism that produces the enzyme. The enzyme can be an unpurified, crude, or purified enzyme. These enzymes may be used as a solid-phase protein immobilized on a solid phase in a reaction. The enzyme used can be used in a form of a transformed microorganism, e.g., a cultured product of the microorganism, that produces the enzyme or a treated product thereof, e.g., a disrupted product, a lysate, or a lyophilizate of the microorganism.

(105) The enzyme used in the method is isolated by a known method, and further purified as necessary to obtain a desired enzyme. As the microorganism that produces the enzyme, a transformed microorganism is preferable from a viewpoint of obtaining a large amount of the enzyme, and the like. The term transformation is intended not only for introduction of a polynucleotide into a host cell but also for modification of a genome in the host cell.

(106) Culture conditions of the transformed microorganism are not particularly limited, and standard cell culture conditions can be used depending on a host. A medium for culturing the transformed microorganism is known, and for example, a carbon source, a nitrogen source, a vitamin source, or the like can be added to a nutrient medium such as an LB medium or a minimum medium such as an M9 medium.

(107) Culture temperature is 4 to 40 C., or 10 to 37 C. Culture time is 5 to 168 hours, or 8 to 72 hours. As a gas composition, a CO.sub.2 concentration is about 6% to about 84%, and a pH of about 5 to 9 is preferable. In addition, culture is performed under aerobic, anoxic, or anaerobic conditions depending on the properties of a host cell.

(108) As a culturing method, any appropriate method can be used. Depending on a host cell, either shaking culture or static culture is possible, but stirring may be performed as necessary, or aeration may be performed. Examples of such a culture method include a batch culture method, a fed-batch culture method, and a continuous culture method. When expression of a specific protein produced by the transformant microorganism is under control of an inducible promoter such as a lac promoter, an inducer such as isopropyl--thiogalactopyranoside (IPTG) may be added to a culture medium to induce the expression of the protein.

(109) The produced target enzyme can be purified and isolated from the extract of the transformed microorganism by known salting out, a precipitation method such as an isoelectric point precipitation method or a solvent precipitation method, a method using a molecular weight difference, such as dialysis, ultrafiltration, or gel filtration, a method using specific affinity, such as ion exchange chromatography, a method using a difference in hydrophobicity, such as hydrophobic chromatography or reverse phase chromatography, affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric point electrophoresis, or the like, or a combination thereof. When the target enzyme is secreted and expressed, a culture supernatant containing the target enzyme can be obtained by removing bacterial cells from a culture solution obtained by culturing the transformed microorganism by centrifugation or the like. The target enzyme can also be purified and isolated from the culture supernatant.

(110) The amino group-containing compound and the carboxyl group-containing compound which are substrates used in the method can be added to a reaction system containing the enzyme, such as e.g. an aqueous solution containing the enzyme, a culture solution containing a transformed microorganism that produces the enzyme, or a treated product of the transformed microorganism that produces the enzyme. Alternatively, in the method, an amino group-containing compound or a carboxyl group-containing compound produced in another reaction system can also be used as a substrate.

(111) When the method is performed using the enzyme itself, such as e.g. purified enzyme, an aqueous solution containing the enzyme can be used as the reaction system. The aqueous solution can be a buffer. Examples of the buffer include a phosphate buffer, a Tris buffer, a carbonate buffer, an acetate buffer, and a citrate buffer. The aqueous solution has a pH of, for example, about 5 to 10. The amounts of the enzyme, and the amino group-containing compound and the carboxyl group-containing compound (substrates) in the reaction system, and reaction time can be appropriately adjusted depending on the amount of the N-acyl-amino group-containing compound to be produced. Reaction temperature is not particularly limited as long as the reaction proceeds, but can be 20 to 40 C.

(112) The method may be performed in combination with an ATP regeneration system. When the method is performed using the enzyme itself, such as e.g. a purified enzyme, examples of the combination with the ATP regeneration system include a reaction by a combination with an ATP regeneration enzyme, such as by e.g. mixing. Examples of the ATP regeneration enzyme include polyphosphate kinase, a combination of polyphosphate: AMP phosphate transferase and polyphosphate kinase, and a combination of polyphosphate: AMP phosphate transferase and adenylate kinase. When the method is performed using a transformed microorganism that produces the enzyme or a treated product thereof, examples of the combination with the ATP regeneration system include use of a microorganism having enhanced ability to supply ATP as a host. Examples of the microorganism having enhanced ability to supply ATP include a microorganism that produces or enhances the above-described ATP regeneration enzyme. Examples of the microorganism that produces or enhances the ATP regeneration enzyme include a host into which an expression unit of the ATP regeneration enzyme is introduced by transformation, a host containing a mutation that enhances the amount of expression of the ATP regeneration enzyme in a host genome, and a host containing a mutation that enhances an activity of the ATP regeneration enzyme in the host genome.

(113) Production of N-Acyl-Amino Group-Containing Compound by Microorganism Culture Method

(114) In the method, the reaction in the presence of the enzyme may be performed using a transformed microorganism, e.g. a cultured product of the microorganism, that produces the enzyme.

(115) When the method is performed using a cultured product of the transformed microorganism, the transformed microorganism may be, for example, a host having enhanced ability to incorporate an amino acid and/or a fatty acid in order to improve supply efficiency of a substrate of an enzymatic reaction to improve production efficiency. Examples of the host having the enhanced incorporating ability include those described above. In this case, by culturing the transformed microorganism in a culture solution 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 incorporated into the transformed microorganism, and an amide bond is formed by an enzyme produced in the transformed microorganism. As a result, a target N-acyl-amino group-containing compound, e.g. an N-acylamino acid, can be produced.

(116) In addition, in order to suppress a loss of the substrate in the enzymatic reaction and/or to promote supply of the substrate in the enzymatic reaction or to suppress a loss of a product, the transformed microorganism may have, for example, at least one of the following genetic modifications: (1) enhancement of ability to supply a fatty acid; (2) enhancement of ability to supply an amino acid; (3) enhancement of ability to supply ATP; and (4) deficiency or attenuation of an N-acylamino acid degrading enzyme.

(117) Such a transformed microorganism may have a genetic modification effective for enhancement of ability to supply a fatty acid, and such a transformed microorganism may be a transformed microorganism in which a fatty acid degradation system is attenuated or deficient and/or its synthesis system is enhanced. Examples of the transformed microorganisms (host) in which the degradation system is attenuated or deficient and/or the synthesis system is enhanced include those described above. Preferable examples of such a transformed microorganism include a microorganism which includes at least one genetic modification: (a) deficiency or attenuation of acyl-CoA synthetase; and (b) enhancement of acyl-ACP thioesterase.

(118) Details of the genetic modifications of (a) and (b) are as described above.

(119) When the N-acyl-amino group-containing compound is produced by culturing the transformed microorganism, culture conditions of the transformed microorganism are not particularly limited, and for example, culture conditions for performing culture in a medium further containing a predetermined amount of an amino group-containing compound and/or carboxyl group-containing compound under the culture conditions described above can be used.

(120) Production of N-Acyl-Amino Group-Containing Compound by Direct Fermentation Method

(121) Described herein is a method for producing an N-acyl-amino group-containing compound by a direct fermentation method. The direct fermentation method 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 solution containing a carbon source as a raw material to produce a target N-acyl-amino group-containing compound, e.g. an N-acylamino acid, from the carbon source. This method is based on a principle that a carbon source is incorporated into a microorganism, the carbon source is metabolized into a carboxyl group-containing compound, e.g. a fatty acid and an amino group-containing compound, e.g. an amino acid in the microorganism, 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 bonded to each other by an amide bond due to an action of an enzyme having an N-acylation activity in the microorganism, thereby producing a target N-acyl-amino group-containing compound, e.g. an N-acylamino acid.

(122) For example, the direct fermentation 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 bonded to each other by an amide bond, the method including culturing, in the presence of a carbon source, a microorganism which includes at least one of the following genetic modifications: (1) enhancement of ability to supply a fatty acid; (2) enhancement of ability to supply an amino acid; (3) enhancement of ability to supply ATP; and (4) deficiency or attenuation of an N-acylamino acid degrading enzyme, and which includes an expression unit of a polynucleotide encoding an enzyme having an N-acylation activity. Details of the above (1) to (4) are similar to those described above (other portions are also similar).

(123) The direct fermentation 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 bonded to each other by an amide bond, the method including culturing, in the presence of a carbon source, a microorganism which includes at least one of the following genetic modifications: (a) deficiency or attenuation of acyl-CoA synthetase; and (b) enhancement of acyl-ACP thioesterase, and which includes an expression unit of a polynucleotide encoding an enzyme having an N-acylation activity. Details of the above (a) and (b) are similar to those described above (other portions are also similar).

(124) Examples of the carbon source include a saccharide, a lipid, a protein, and an alcohol, e.g. glycerin, and a saccharide is preferable. Examples of the saccharide include glucose, galactose, mannose, fructose, sucrose, maltose, lactose, starch hydrolysate, and molasses, and glucose is a particular example.

(125) Details of the genetic modifications of (a) and (b) are as described above. At least one genetic modification of (a) and (b) suppresses a loss of a substrate in the enzymatic reaction and/or promotes supply of the substrate in the enzymatic reaction, thereby enhancing the metabolism from the carbon source to the carboxyl group-containing compound, e.g. a fatty acid in the microorganism.

(126) An amino group-containing compound, e.g. an amino acid, and a carboxyl group-containing compound, e.g. a fatty acid, produced by metabolism are bonded to each other by an amide bond due to an action of an enzyme having an N-acylation activity to produce an N-acyl-amino group-containing compound, e.g. an N-acylamino acid, as a target product.

(127) The enzyme having an N-acylation activity refers to an enzyme having ability to bond a carboxyl group and an amino group to each other in an ATP-dependent manner to form an amide bond. As the enzyme having an N-acylation activity, the above-described modified enzyme having an N-acylation activity, such as the modified enzyme as described herein, may be used, and a protein found to have ability to bond a carboxyl group and an amino group to each other in an ATP-dependent manner to form an amide bond, a protein containing an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids in an amino acid sequence of the protein and having an N-acylation activity, and a protein containing an amino acid sequence having 90% or more identity to an amino acid sequence of the protein and having an N-acylation activity may be used. Examples of the protein found to have ability to bond a carboxyl group and an amino group to each other in an ATP-dependent manner to form an amide bond include a GH3 protein, such as AtGH3-6, OsGH3-8, AtJAR1 (AtGH3-11), AtGH3-5, AtGH3-10, AtGH3-12, AtGH3-17, SsGH3, or CfHP (WP_002626336)) and a PaaK protein (PsIAAL or PaHP (WP_031591948)) described in PCT/JP2019/007681. AtGH3-6 (SEQ ID NO: 1) may be used. The GH3 protein and the PaaK protein also include mutants thereof, such as a protein containing an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids in an amino acid sequence of the protein and having an N-acylation activity, and a protein containing an amino acid sequence having 90% or more identity to an amino acid sequence of the protein and having an N-acylation activity.

(128) Examples of the amino group-containing compound produced by metabolism include those described above as examples of the amino group-containing compound, but an amino acid is a particular example, and L-glutamic acid or L-aspartic acid is further examples. Examples of the carboxyl group-containing compound produced by metabolism include those described above as examples of the carboxyl group-containing compound, but a fatty acid, such as e.g. a saturated fatty acid, a fatty acid having 8 to 18 carbon atoms, e.g. a saturated fatty acid, a fatty acid having 12 carbon atoms, and lauric acid.

(129) When the N-acyl-amino group-containing compound is produced by culturing the transformed microorganism, culture conditions of the transformed microorganism are not particularly limited, and for example, culture conditions for performing culture in a medium further containing a predetermined amount of a carbon source, e.g. a saccharide such as glucose, can be used.

(130) Production of the N-acyl-amino group-containing compound can be appropriately confirmed. For example, such confirmation can be performed by adding a reaction stop solution, e.g. an aqueous solution of 1.4% (w/v, phosphoric acid and 75% (v/v, methanol, to the reaction system and subjecting the supernatant after centrifugation to UPLC-MS analysis.

(131) Surfactant

(132) The N-acyl-amino group-containing compound, e.g. an N-acyl-amino group-containing compound produced by the method, or a salt thereof may be used for various applications, and for example, may be used as a component contained in a composition such as a cosmetic material, e.g. a surfactant. The N-acyl-amino group-containing compound or a salt thereof contained in such a composition may contain, for example, an N-monounsaturated acyl-amino group-containing compound or a salt thereof. Examples of the N-monounsaturated acyl-amino group-containing compound or a salt thereof include those described above.

(133) When the N-acyl-amino group-containing compound or a salt thereof is used as a component of a surfactant, the surfactant may include an N-monounsaturated acyl-amino group-containing compound having a monounsaturated acyl group having 10 to 16 carbon atoms or a salt thereof. In such a case, since an acyl moiety of the N-monounsaturated acyl-amino group-containing compound is a monounsaturated acyl, an effect that the N-monounsaturated acyl-amino group-containing compound suppresses an increase in viscosity, has a low viscosity with high handleability, and has good solubility is exhibited. In addition, since the acyl moiety has 10 to 16 carbon atoms, good foaming and foam quality are obtained.

(134) Examples of the salt of the N-acyl-amino group-containing compound include those described above.

(135) When the N-acyl-amino group-containing compound is obtained from a monounsaturated fatty acid, the N-acyl-amino group-containing compound may be that obtained, for example, by reacting a monounsaturated fatty acid derivative represented by C.sub.n-1H.sub.mCOX, wherein X represents any monovalent group, for example, a halogen atom selected from the group consisting of fluorine, chlorine, bromine, and iodine, with an acidic amino acid salt. Examples of the salt include inorganic salts and organic salts as described above.

(136) Examples of the monounsaturated fatty acid from which the N-monounsaturated acyl-amino group-containing compound is derived include 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.

(137) 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.

(138) 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).

(139) The N-monounsaturated acyl-amino group-containing compound may include one kind of N-monounsaturated acyl-amino group-containing compound, or two or more kinds of N-monounsaturated acyl-amino group-containing compound. That is, the N-monounsaturated acyl-amino group-containing compound may include one or more 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 kinds of N-monounsaturated acyl-amino group-containing compound are used, a combination of N-dodecenoyl acidic amino acid (C12: 1) and one or more of N-decenoyl acidic amino acid (C10: 1), N-tetradecenoyl acidic amino acid (C14: 1), and N-hexadecenoyl acidic amino acid (C16: 1) is preferable, and a combination of N-dodecenoyl acidic amino acid (C12: 1) and N-hexadecenoyl acidic amino acid (C16: 1) is more preferable from the viewpoint of improving foam quality.

(140) A unsaturated bond site of the N-monounsaturated acyl acidic amino acid is not particularly limited, and the unsaturated bond may be present at a carbon chain terminal of an acyl moiety, between carbon atoms separated by several carbon atoms from the carbon chain terminal of the acyl moiety, or at an -position of a carbonyl group of the acyl moiety. In particular, the unsaturated bond can be present between carbon atoms separated by 6 or 7 carbon atoms from the carbon chain terminal of the acyl moiety. That is, the N-monounsaturated acyl acidic amino acid is a compound represented by the following general formula (A).

(141) ##STR00002##

(142) In general formula (A), 1 is an integer of 1 or 2, and m is an integer of 0 to 6.

(143) The stereochemistry (unsaturated double bond site) of the N-monounsaturated acyl acidic amino acid may be either in a cis or trans conformation. However, a cis conformation is preferable from a viewpoint of suppressing an increase in viscosity and achieving a low viscosity with high handleability.

(144) Composition

(145) The composition includes component (A) an N-monounsaturated acyl acidic amino acid or a salt thereof, wherein the unsaturated acyl is an acyl having 10 to 16 carbon atoms, and component (B) an N-saturated acyl acidic amino acid or a salt thereof.

(146) The composition includes component (A), and therefore has good foaming action, foam quality, and solubility even when weakly acidic having a pH equivalent to that of the skin, and has a low viscosity with high handleability. In addition, the composition is a composition also including component (B) and having variations in an acyl group, and therefore has an effect of improving oil cleansing power.

(147) Component (A)

(148) Details of component (A) are as described in the N-monounsaturated acyl acidic amino acid of the surfactant.

(149) Component (B)

(150) The N-saturated acyl acidic amino acid refers to a compound represented by C.sub.n-1H.sub.mCONHCHRCOOH, in which the hydrogen atom represented by H.sub.m may be replaced, m is as defined above, and R represents a side chain of an acidic amino acid, as a free form. That is, the N-saturated acyl acidic amino acid refers to a compound in which one hydrogen atom on an amino group of an acidic amino acid is replaced with a saturated acyl group. The N-saturated acyl acidic amino acid may be a free form of an N-saturated acyl acidic amino acid or a salt of the N-saturated acyl acidic amino acid.

(151) Note that the same as the salt of the N-monounsaturated acyl acidic amino acid applies to the salt of the N-saturated acyl acidic amino acid.

(152) The N-saturated acyl acidic amino acid may be derived from a fatty acid represented by C.sub.n-1H.sub.mCOOH or a derivative thereof for convenience. In addition, a saturated acyl having n carbon atoms and a fatty acid and the like from which the saturated acyl having n carbon atoms is derived may be represented by Cn. For example, N-lauroyl acidic amino acid (C12, refers to a compound in which one hydrogen atom on an amino group of an acidic amino acid is replaced with a lauroyl group which is an acyl group derived from lauric acid (C12), C.sub.11H.sub.23CONHCHRCOOH in which R represents a side chain of the acidic amino acid.

(153) When the N-saturated acyl acidic amino acid is obtained from a saturated fatty acid, the N-saturated acyl acidic amino acid can be obtained, for example, by reacting a saturated fatty acid derivative represented by C.sub.n-1H.sub.mCOX, wherein X represents any monovalent group, for example, a halogen atom of fluorine, chlorine, bromine, and iodine, with an acidic amino acid or a salt thereof. Examples of the salt include inorganic salts and organic salts as described above.

(154) Examples of the N-saturated acyl acidic amino acid 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).

(155) The acidic amino acid in the N-saturated acyl acidic amino acid refers to an amino acid having an acidic side chain. Examples of the acidic amino acid include glutamic acid and aspartic acid.

(156) As the N-saturated acyl acidic amino acid, 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) is examples.

(157) Component (B) may include one kind of N-saturated acyl acidic amino acid or a salt thereof, or two or more kinds of N-saturated acyl acidic amino acids or salts thereof. That is, component (B) may include one or more 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 component (B) includes two or more kinds of N-saturated acyl acidic amino acids, component (B) can include N-lauroyl acidic amino acid (C12), or includes N-lauroyl acidic amino acid (C12) and one or more 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) in combination.

(158) When component (B) includes N-lauroyl acidic amino acid (C12), the content of N-lauroyl acidic amino acid (C12) in component (B) is, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, or 60% by mass or more. More specifically, the content of N-lauroyl acidic amino acid in component (B) is, for example, 30 to 100% by mass, 40 to 100% by mass, 50 to 100% by mass, or 60 to 100% by mass.

(159) A mass ratio of component (A) to the total of component (A) and component (B) (A/(A+B)) is usually 0.001 or more, 0.002 or more, 0.003 or more, or 0.004 or more from the viewpoint of improving solubility at a low pH by component (A), and the like. The mass ratio of component (A) to the total of component (A) and component (B) (A/(A+B)) is usually 1.00 or less, less than 1.00, 0.80 or less, 0.60 or less, or 0.50 or less from the viewpoint of contribution to oil cleansing power due to a large content of component (B), and the like. 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, 0.001 or more and less than 1.00, 0.002 to 0.80, 0.003 to 0.60, or 0.004 to 0.50.

(160) The composition may further include component (C) an N-unsaturated fatty acid or a salt thereof.

(161) When the composition includes component (C), the foam quality is better, and the oil cleansing power is further improved.

(162) Component (C)

(163) The number of carbon atoms of the unsaturated fatty acid can be 6 to 22, or 8 to 18. Examples of the unsaturated fatty acid 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).

(164) Examples of the salt of the unsaturated fatty acid include an inorganic salt such as a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or an aluminum salt; an organic amine salt such as an ammonium salt, a monoethanolamine salt, a diethanolamine salt, or a triethanolamine salt; and an organic salt such as a basic amino acid salt including an arginine salt and a lysine salt. Among these salts, a triethanolamine salt, a sodium salt, and a potassium salt are particular examples.

(165) Component (C) may include one kind of unsaturated fatty acid or a salt thereof, or two or more kinds of unsaturated fatty acids or salts thereof. That is, component (C) may include one or more 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 component (C) includes two or more kinds of unsaturated fatty acids or salts thereof, component (C) includes dodecenoic acid (C12: 1), and can include dodecenoic acid (C12: 1) and one or more of octenoic acid (C8: 1), decenoic acid (C10: 1), tetradecenoic acid (C14: 1), and hexadecenoic acid (C16: 1) in combination.

(166) When component (C) includes dodecenoic acid (C12: 1), the amount of dodecenoic acid (C12: 1) in component (C) is, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, or 80% by mass or more. More specifically, the amount of dodecenoic acid (C12: 1) in component (C) is, for example, 30 to 100% by mass, 40 to 100% by mass, 50 to 100% by mass, 60 to 100% by mass, 70 to 100% by mass, or 80 to 100% by mass.

(167) A mass ratio of component (C) to the total of component (A) and component (B) (C/(A+B)) may be 0 or more. When the mass ratio of component (C) to the total of component (A) and component (B) (C/(A+B)) is 0, component (C) is not present. The mass ratio can be 0.01 or more, and can be 0.1 or more, 0.2 or more, 0.4 or more, or 0.5 or more from the viewpoint of contribution of component (C) to improvement of foam quality (density), and the like. The mass ratio of component (C) to the total of component (A) and component (B) (C/(A+B)) is usually 20 or less, 15 or less, 13 or less, 11 or less, or 10 or less from the viewpoint of contribution of an effect of component (A) and component (B), and the like. 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, 0.1 to 15, 0.4 to 11, or 0.5 to 10.

(168) The composition may include component (A), component (B), and an optional component (C) in (D) a water-soluble medium. As the water-soluble medium, any water-soluble solvent can be used. Examples of the water-soluble medium include an aqueous solution. The aqueous solution may have buffering ability or does not have to have buffering ability. Examples of the aqueous solution include water, e.g. distilled water, sterilized distilled water, purified water, physiological saline, or tap water such as city water, a phosphoric acid buffer, a Tris-hydrochloric acid buffer, a TE (Tris-EDTA, buffer, a carbonic acid buffer, a boric acid buffer, a tartaric acid buffer, a glycine buffer, a citric acid buffer, and an acetic acid buffer.

(169) The amount of component (A) varies depending on various conditions such as the kinds and concentrations of other components included in the composition and a pH, and therefore is not particularly limited. When the composition is in a form of an aqueous solution, the amount of component (A) is, for example, 0.001% by mass or more, 0.002% by mass or more, and 0.003% by mass or more from the viewpoint of improving solubility at a low pH, and the like. The amount of component (A) can be, for example, 60% by mass or less, and can be 40% by mass or less. More specifically, the amount of component (A) is, for example, 0.001 to 60% by mass, 0.001 to 40% by mass, 0.002 to 40% by mass, and 0.003 to 40% by mass.

(170) The content of component (B) varies depending on various conditions such as the kinds and concentrations of other components included in the composition and the pH, and therefore is not particularly limited. When the composition is in a form of an aqueous solution, the content of component (B) is, for example, 0.01% by mass or more, 0.02% by mass or more, and 0.03% by mass or more from a viewpoint of improving solubility at a low pH, and the like. The amount of component (B) can be, for example, 60% by mass or less, 40% by mass or less, or 10% by mass or less. More specifically, the amount of component (B) is, for example, 0.01 to 60% by mass, 0.02 to 40% by mass, or 0.03 to 10% by mass.

(171) The amount of component (C) varies depending on various conditions such as the kinds and concentrations of other components included in the composition and the pH, and therefore is not particularly limited, but may be 0% by mass or more. When the amount of component (C) is 0% by mass, component (C) is not present. When the composition is in a form of an aqueous solution, the amount of component (C) is, for example, 0.001% by mass or more, 0.002% by mass or more, and 0.003% by mass or more from the viewpoint of contribution to improvement of foam quality (density), and the like. The content of component (C) can be, for example, 5% by mass or less, 3% by mass or less, or 1% by mass or less from the viewpoint of inhibiting the effect when the component (C) is included in a large amount, and the like. More specifically, the amount of component (C) is, for example, 0.001 to 5% by mass, 0.002 to 3% by mass, or 0.003 to 1% by mass.

(172) The composition is weakly acidic from the viewpoint of storage stability due to suppression of growth of various bacteria (antiseptic effect) and low irritation to the skin due to a pH equivalent to that of the skin (weakly acidic). The pH of the composition is, for example, 3.0 to 9.0 and can be 3.0 to 8.0 or 3.0 to 7.0. An upper limit of the pH is 8.0 or less, or 7.0 or less. In addition, a lower limit is 4.0 or more, or 4.5 or more. The pH of the composition is 4.0 to 8.0, 4.0 to 7.0, 4.5 to 8.0, or 4.5 to 7.0 from the viewpoint of achieving a pH equivalent to that of the skin. The composition has excellent solubility even at a low pH, and therefore suppresses precipitation. It can be said that the composition has excellent storage stability also in this respect. Furthermore, in general, when the pH of an anionic surfactant decreases, foaming tends to decrease. However, the composition has good foaming even at a low pH. The pH can be adjusted using a pH adjusting agent. Examples of the pH adjusting agent include an aqueous solution (buffer) as described above, an acidic substance, e.g. hydrochloric acid, sulfuric acid, nitric acid, or citric acid, and an alkaline substance, e.g. a hydroxide of an alkali metal including sodium and potassium or an alkaline earth metal including calcium.

(173) The composition has a low viscosity from a viewpoint of handleability. The viscosity of the composition is, for example, 1.5 Pas or less, 1.0 Pa's or less, 0.80 Pa's or less, 0.70 Pa's or less, or 0.50 Pa's or less. The composition has excellent handleability, and therefore can be said to be useful as a cosmetic as a personal care.

(174) The viscosity is a value obtained by measuring an aqueous solution of a composition having a concentration of 10% by mass with a B-type viscometer (DVB-10: B-type viscometer manufactured by Toyo Seiki Seisaku-sho, Ltd., rotor Nos. 20 to 23, 6 to 30 rpm, 25 C., after 30 seconds).

(175) The composition may also include another component such as an additional cleansing component, a polyhydric alcohol, a thickener, a stabilizer, a preservative, a fragrance, or a pigment. Specific kinds and amounts of these components can be appropriately set.

(176) Examples of the additional cleansing component include a surfactant such as an anionic surfactant, an amphoteric surfactant, or a nonionic surfactant, and a microsolid, e.g. a microsphere or a scrub.

(177) The anionic surfactant includes one or more anionic groups. Examples of the anionic group include a carboxyl group, a sulfonic acid group, a sulfuric acid group, and a phosphoric acid group. Examples of the anionic surfactant include a higher fatty acid, an N-acylamino acid, an N-acyltaurine, an alkyl ether carboxylic acid, an alkyl phosphoric acid, a polyoxyethylene alkyl ether phosphoric acid, an alkyl sulfuric acid, a polyoxyethylene alkyl ether sulfuric acid, a sulfonic acid compound having an alkyl chain, and salts thereof.

(178) The amphoteric surfactant includes 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 amide betaine amphoteric surfactant, a betaine acetate amphoteric surfactant, a sulfobetaine amphoteric surfactant, and an imidazoline amphoteric surfactant, e.g. lauroamphoacetic acid or a salt thereof.

(179) Examples of the nonionic surfactant include an ester type surfactant such as a glycerin fatty acid ester, a sorbitan fatty acid ester, or a sucrose fatty acid ester, an ether type surfactant such as an alkyl polyethylene glycol or a polyoxyethylene alkyl phenyl ether, and a nonionic surfactant such as an alkyl glycoside in which a saccharide and a higher alcohol are bonded to each other by a glycosidic bond or an alkyl polyglycoside.

(180) Examples of the polyhydric alcohol include a dihydric alcohol, 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, or monoglyceride such as monoacylglycerol, a trihydric alcohol, e.g. glycerin, trimethylolpropane, or 1,2,6-hexanetriol, a tetrahydric alcohol, e.g. diglycerin or pentaerythritol, an alcohol having a higher valence, and salts thereof, e.g. inorganic salts and organic salts as described above. Examples of the alcohol having a higher valence include a sugar alcohol optionally having a substituent. e.g. a monosaccharide alcohol such as sorbitol, mannitol, sucrose, glucose, or mannose, a disaccharide alcohol such as trehalose, and a polysaccharide alcohol such as hyaluronic acid or xanthan gum, a polymer of a dihydric to tetrahydric alcohol as described above, e.g. polyglycol or polyglycerin, and salts thereof, e.g. inorganic salts and organic salts as described above. The polyhydric alcohol is a dihydric to tetrahydric alcohol, or a dihydric or trihydric alcohol.

(181) Examples of the thickener include carrageenan, dextrin, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyacrylic acid, polymethacrylic acid, carboxyvinyl polymer such as carbomer, acrylic acid/alkyl acrylate (C10-30, copolymer, and xanthan gum.

(182) Examples of the stabilizer include ascorbic acid, sodium pyrosulfite, and EDTA.

(183) Examples of the preservative include ethyl parahydroxybenzoate, sodium benzoate, salicylic acid, sorbic acid, paraben, such as methylparaben, propylparaben, or the like, and sodium bisulfite.

(184) Examples of the fragrance include a natural fragrance and a synthetic fragrance. Examples of the natural fragrance include rose oil, jasmine oil, neroli oil, lavender oil, ylang-ylang oil, tubellows oil, clary sage oil, clove oil, peppermint oil, geranium oil, patulie oil, sandalwood oil, cinnamon oil, coriander oil, nutmeg oil, pepper oil, lemon oil, orange oil, bergamot oil, opoponax oil, vetiver oil, oris oil, and oak moss oil. Examples of the synthetic fragrance include limonene (orange), -caryophyllene (woody), cis-3-hexenol (young green leaves), linalool (lily of the valley), farnesol (floral with fresh green notes), -phenylethyl alcohol (rose), 2,6-nonadienal (violet or cucumber), citral (lemon), -hexyl cinnamic aldehyde (jasmine), -ionone (violet when being diluted), t-carboxylic (spearmint), cyclopentadecanone (musk), linalyl acetate (bergamot or lavender), benzyl benzoate (balsam), -undecalactone (peach), eugenol (clove), rose oxide (green floral), indole (jasmine when being diluted), phenylacetaldehyde dimethyl acetal (hyacinth), auranthiol (orange flower), and menthol (peppermint). The word in the parentheses indicates an aroma.

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

(186) The composition can be provided in various forms such as powder, liquid, gel, paste, cream, and foam. Note that the composition can be produced by a usual method.

(187) The composition can be a cosmetic in any form applicable to, for example, the skin, hair, or scalp according to a usual method. The cosmetic is suitable for applications such as a body shampoo, a hand soap, a facial cleanser, a cleansing lotion, a cleansing cream, a massage cream, and a hair shampoo as a cleanser for animals such as humans. Properties of the cosmetic, e.g. pH, is similar to that of the composition described above.

(188) In addition, the composition can also be used as an additive such as an excipient.

EXAMPLES

(189) The present invention will be described in more detail by describing the following Examples, but the present invention is not limited to the following Examples.

Example 1: Construction of Mutant AtGH3-6 Expression Plasmid by Site-Directed Mutagenesis

(190) (1) A mutation was introduced into AtGH3-6 using, as a template, pET-28a-AtGH3-6 (PCT/JP2019/007681) that expresses Arabidopsis thaliana-derived indole-3-acetic acid-amido synthetase GH3.6 (AtGH3-6, Q9LSQ4, SEQ ID NO: 1 for an amino acid sequence, SEQ ID NO: 2 for a nucleotide sequence encoding the amino acid sequence in which a codon is optimized for expression in E. coli) in which a His-tag and a thrombin recognition sequence were fused to an N-terminal side. In addition, into some samples of mutant AtGH3-6, a mutation was further introduced using, as a template, a mutant AtGH3-6 expression plasmid obtained below. A method for constructing an expression plasmid of mutant AtGH3-6 (Mutant No. 31) is described in Example 1 (2). Mutagenesis was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) under the following conditions. 1 cycle 98 C., 30 sec 30 cycles 98 C., 10 sec 55 C., 15 sec 72 C., 40 sec 1 cycle 72 C., 5 min 4 C., hold Primers used are as follows. WT in Table indicates wild type AtGH3-6.

(191) TABLE-US-00002 TABLE2-1 Primerforsite-directedmutagenesisusedinpreparation ofmutantAtGH3-6(1) Introduced SEQ Mutant mutation ID No. Template point Nucleotidesequence(5to3) NO R18 WT Y134F CGTCGGAGTCTGCTGTTTTCACTCCTTAT 7 GCCC GGGCATAAGGAGTGAAAACAGCAGACT 8 CCGACG R20 WT Y134V CGTCGGAGTCTGCTGGTGTCACTCCTTAT 9 GCCC GGGCATAAGGAGTGACACCAGCAGACT 10 CCGACG R30 WT S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 11 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 12 AGGAA R32 WT V174A CTGCCTGCACGTCCCGCGCTGACCTCGT 13 ATTAC GTAATACGAGGTCAGCGCGGGACGTGCA 14 GGCAG R39 WT V231A CTGCGTGTTGGCGCGGCGTTTGCCAGCG 15 GGTTT AAACCCGCTGGCAAACGCCGCGCCAACA 16 CGCAG R55 WT T336S TTACCGTTAGTCTGCAGCATGTATGCGA 17 GCAGT ACTGCTCGCATACATGCTGCAGACTAAC 18 GGTAA R56 WT M337G CCGTTAGTCTGCACGGGCTATGCGAGCA 19 GTGAA TTCACTGCTCGCATAGCCCGTGCAGACT 20 AACGG R59 WT A339G GTCTGCACGATGTATGGCAGCAGTGAAT 21 GCTAC GTAGCATTCACTGCTGCCATACATCGTG 22 CAGAC R61 WT S340A TGCACGATGTATGCGGCGAGTGAATGCT 23 ACTTT AAAGTAGCATTCACTCGCCGCATACATC 24 GTGCA R68 WT Y344G GCGAGCAGTGAATGCGGCTTTGGAGTGA 25 ATCTC GAGATTCACTCCAAAGCCGCATTCACTG 26 CTCGC R69 WT Y344A GCGAGCAGTGAATGCGCGTTTGGAGTGA 27 ATCTC GAGATTCACTCCAAACGCGCATTCACTG 28 CTCGC R70 WT Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 29 ATCTC GAGATTCACTCCAAACACGCATTCACTG 30 CTCGC R78 WT S455T GTTCTCTCCATTGACACCGACAAGACGG 31 ATGAA TTCATCCGTCTTGTCGGTGTCAATGGAG 32 AGAAC R1002 WT T122S/ CGCAAACTTATGCCGAGCACCGAGGAAG 33 I123T AACTGGAT ATCCAGTTCTTCCTCGGTGCTCGGCATAA 34 GTTTGCG R1005 WT R117P ACCTCAGGCGGAGAACCGAAACTTATGC 35 CGACG CGTCGGCATAAGTTTCGGTTCTCCGCCTG 36 AGGT R117P Q533R AATAGCGTGTATCGCCGTGGCCGTGTGT 37 CCGAC GTCGGACACACGGCCACGGCGATACACG 38 CTATT

(192) TABLE-US-00003 TABLE2-2 Primerforsite-directedmutagenesisusedinpreparationof mutantAtGH3-6(2) Introduced Mutant mutation SEQID No. Template point Nucleotidesequence(5to3) NO 4 WT C576A TACAAAACACCAAGAGCGGTCAAATTCG 39 CGCCG CGGCGCGAATTTGACCGCTCTTGGTGTTT 40 TGTA 7 R32 S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 41 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 42 AGGAA 8 R39 S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 43 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 44 AGGAA 9 R56 S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 45 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 46 AGGAA 10 R70 S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 47 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 48 AGGAA 11 R39 V174A CTGCCTGCACGTCCCGCGCTGACCTCGT 49 ATTAC GTAATACGAGGTCAGCGCGGGACGTGCA 50 GGCAG 12 R32 M337G CCGTTAGTCTGCACGGGCTATGCGAGCA 51 GTGAA TTCACTGCTCGCATAGCCCGTGCAGACT 52 AACGG 13 R32 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 53 ATCTC GAGATTCACTCCAAACACGCATTCACTG 54 CTCGC 14 R39 M337G CCGTTAGTCTGCACGGGCTATGCGAGCA 55 GTGAA TTCACTGCTCGCATAGCCCGTGCAGACT 56 AACGG 15 R39 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 57 ATCTC GAGATTCACTCCAAACACGCATTCACTG 58 CTCGC 16 R56 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 59 ATCTC GAGATTCACTCCAAACACGCATTCACTG 60 CTCGC 17 R1005 S161P TTCCTCTTCATCAAGCCGGAATCGAAAA 61 CCCCT AGGGGTTTTCGATTCCGGCTTGATGAAG 62 AGGAA 18 R1005 V174A CTGCCTGCACGTCCCGCGCTGACCTCGT 63 ATTAC GTAATACGAGGTCAGCGCGGGACGTGCA 64 GGCAG 19 R1005 V231A CTGCGTGTTGGCGCGGCGTTTGCCAGCG 65 GGTTT AAACCCGCTGGCAAACGCCGCGCCAACA 66 CGCAG 20 R1005 M337G CCGTTAGTCTGCACGGGCTATGCGAGCA 67 GTGAA TTCACTGCTCGCATAGCCCGTGCAGACT 68 AACGG 21 R1005 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 69 ATCTC GAGATTCACTCCAAACACGCATTCACTG 70 CTCGC 24 WT Y344I GAATGCATTTTTGGAGTGAATCTCAGG 71 TCCAAAAATGCATTCACTGCTCGCATA 72

(193) TABLE-US-00004 TABLE2-3 Primerforsite-directedmutagenesisusedinpreparation ofmutantAtGH3-6(3) Introduced Mutant mutation SEQID No. Template point Nucleotidesequence(5to3) NO 30 18 V231A CTGCGTGTTGGCGCGGCGTTTGCCAGCG 73 GGTTT AAACCCGCTGGCAAACGCCGCGCCAACA 74 CGCAG 32 18 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 75 ATCTC GAGATTCACTCCAAACACGCATTCACTG 76 CTCGC 34 19 Y344V GCGAGCAGTGAATGCGTGTTTGGAGTGA 77 ATCTC GAGATTCACTCCAAACACGCATTCACTG 78 CTCGC 37 31 G379D GTCCATCGTAACTCAGACGTAACTAGCA 79 GCATT AATGCTGCTAGTTACGTCTGAGTTACGA 80 TGGAC 31+ R350T TTTGGAGTGAATCTCACGCCACTATGCA 81 G379D AACCA TGGTTTGCATAGTGGCGTGAGATTCACT 82 CCAAA 38 31 V311A ACCAAATATGTTGACGCGATTGTCACTG 83 GCACC GGTGCCAGTGACAATCGCGTCAACATAT 84 TTGGT 39 31 L137I CTGCTGTACTCACTCATTATGCCCGTCAT 85 GGAT ATCCATGACGGGCATAATGAGTGAGTAC 86 AGCAG 40 31 Q200E TATACCAGTCCGAATGAGACGATCTTGT 87 GTTCC GGAACACAAGATCGTCTCATTCGGACTG 88 GTATA 42 31 I123T AAACTTATGCCGACGACCGAGGAAGAA 89 CTGGAT ATCCAGTTCTTCCTCGGTCGTCGGCATAA 90 GTTT 43 31 N101S CAGGTTCTGTGTTCGAGCCCGATAAGCG 91 AGTTT AAACTCGCTTATCGGGCTCGAACACAGA 92 ACCTG 44 40 I123T AAACTTATGCCGACGACCGAGGAAGAA 93 CTGGAT ATCCAGTTCTTCCTCGGTCGTCGGCATAA 94 GTTT 45 40 N101S CAGGTTCTGTGTTCGAGCCCGATAAGCG 95 AGTTT AAACTCGCTTATCGGGCTCGAACACAGA 96 ACCTG 46 42 N101S CAGGTTCTGTGTTCGAGCCCGATAAGCG 97 AGTTT AAACTCGCTTATCGGGCTCGAACACAGA 98 ACCTG 48 37 Q200E TATACCAGTCCGAATGAGACGATCTTGT 99 GTTCC GGAACACAAGATCGTCTCATTCGGACTG 100 GTATA 49 37 I123T AAACTTATGCCGACGACCGAGGAAGAA 101 CTGGAT ATCCAGTTCTTCCTCGGTCGTCGGCATAA 102 GTTT 50 37 N101S CAGGTTCTGTGTTCGAGCCCGATAAGCG 103 AGTTT AAACTCGCTTATCGGGCTCGAACACAGA 104 ACCTG 54 49 N101S CAGGTTCTGTGTTCGAGCCCGATAAGCG 105 AGTTT AAACTCGCTTATCGGGCTCGAACACAGA 106 ACCTG 55 49 Q200E TATACCAGTCCGAATGAGACGATCTTGT 107 GTTCC GGAACACAAGATCGTCTCATTCGGACTG 108 GTATA 56 49 C576A TACAAAACACCAAGAGCGGTCAAATTCG 109 CGCCG CGGCGCGAATTTGACCGCTCTTGGTGTTT 110 TGTA

(194) The obtained PCR product was digested with DpnI. Thereafter, E. coli JM109 was transformed with the reaction solution, and a target plasmid was extracted from a kanamycin resistant strain. This plasmid was defined as a mutant AtGH3-6 expression plasmid.

(195) (2) A mutation was introduced into AtGH3-6 using pET-28a-AtGH3-6 (PCT/JP2019/007681) as a template. In addition, into some samples of mutant AtGH3-6, a mutation was further introduced using, as a template, a mutant AtGH3-6 expression plasmid obtained above or below. Mutagenesis was performed using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) under the following conditions. 1 cycle 98 C., 30 sec 30 cycles 98 C., 10 sec 60 C., 15 sec 68 C., 7.5 min 1 cycle 72 C., 5 min 4 C., hold Primers used are as follows.

(196) TABLE-US-00005 TABLE3 Primersforsite-directedmutagenesisusedinpreparationof mutantAtGH3-6 Introduced Mutant mutation SEQID No. Template point Nucleotidesequence(5to3) NO 26 18 S161P TTCCTCTTCATCAAGCCGGAATCGAA 111 AACCCCT AGGGGTTTTCGATTCCGGCTTGATGA 112 AGAGGAA 27 19 S161P TTCCTCTTCATCAAGCCGGAATCGAA 113 AACCCCT AGGGGTTTTCGATTCCGGCTTGATGA 114 AGAGGAA 29 21 S161P TTCCTCTTCATCAAGCCGGAATCGAA 115 AACCCCT AGGGGTTTTCGATTCCGGCTTGATGA 116 AGAGGAA 31 18 M337G CCGTTAGTCTGCACGGGCTATGCGAG 117 CAGTGAA TTCACTGCTCGCATAGCCCGTGCAGA 118 CTAACGG 33 19 M337G CCGTTAGTCTGCACGGGCTATGCGAG 119 CAGTGAA TTCACTGCTCGCATAGCCCGTGCAGA 120 CTAACGG 41 31 K388N TAGCTTGCCTAATGCACTGACCG 121 CGGTCAGTGCATTAGGCAAGCTA 122 47 31 C576A TACAAAACACCAAGAGCGGTCAAATT 123 CGCGCCG CGGCGCGAATTTGACCGCTCTTGGTGT 124 TTTGTA 57 49 K388N TAGCTTGCCTAATGCACTGACCG 125 CGGTCAGTGCATTAGGCAAGCTA 126 58 56 N101S CAGGTTCTGTGTTCGAGCCCGATAAG 127 CGAGTTT AAACTCGCTTATCGGGCTCGAACACA 128 GAACCTG 59 56 Q200E TATACCAGTCCGAATGAGACGATCTT 129 GTGTTCC GGAACACAAGATCGTCTCATTCGGAC 130 TGGTATA

(197) The obtained PCR product was digested with DpnI. Thereafter, E. coli JM109 was transformed with the reaction solution, and a target plasmid was extracted from a kanamycin resistant strain. This plasmid was defined as a mutant AtGH3-6 expression plasmid. As a result of confirming the sequence, a mutation of M337A was also introduced into Mutant No. 26 in addition to the introduced mutation point.

(198) (3) A mutation was further introduced into AtGH3-6 using pET-28a-mutant AtGH3-6 (Mutant No. 56) as a template. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) under the following conditions. 1 cycle 98 C., 30 sec 30 cycles 98 C., 10 sec 60 C., 15 sec 72 C., 5 sec/kb 1 cycle 72 C., 5 min 4 C., hold Primers used are as follows.

(199) TABLE-US-00006 TABLE4 Primersforsite-directedmutagenesisusedinpreparationof mutantAtGH3-6 Introduced SEQ MutantNo. mutationpoint PCR Nucleotidesequence(5to3) IDNO 60 K388N 1 CGACGACCGAGGAAGAACTGGATCG 131 TCG TCAGTGCATTAGGCAAGCTAATGCTG 132 CT 2 TGCCTAATGCACTGACCGAAAAAGA 133 ACA CTTCCTCGGTCGTCGGCATAAGTTTG 134 CG 61 N101S/Q200E 1 GTTCGAGCCCGATAAGCGAGTTTCTC 135 AC AGATCGTCTCATTCGGACTGGTATAG 136 TT 2 CGAATGAGACGATCTTGTGTTCCGAC 137 TC TTATCGGGCTCGAACACAGAACCTG 138 CGA 62 N101S/K388N 1 GTTCGAGCCCGATAAGCGAGTTTCTC 139 AC TCAGTGCATTAGGCAAGCTAATGCTG 140 CT 2 TGCCTAATGCACTGACCGAAAAAGA 141 ACA TTATCGGGCTCGAACACAGAACCTG 142 CGA 63 Q200E/K388N 1 CGAATGAGACGATCTTGTGTTCCGAC 143 TC TCAGTGCATTAGGCAAGCTAATGCTG 144 CT 2 TGCCTAATGCACTGACCGAAAAAGA 145 ACA AGATCGTCTCATTCGGACTGGTATAG 146 TT 64 N101S/Q200E/ 1 GTTCGAGCCCGATAAGCGAGTTTCTC 147 K388N AC AGATCGTCTCATTCGGACTGGTATAG 148 TT 2 CGAATGAGACGATCTTGTGTTCCGAC 149 TC TCAGTGCATTAGGCAAGCTAATGCTG 150 CT 3 TGCCTAATGCACTGACCGAAAAAGA 151 ACA TTATCGGGCTCGAACACAGAACCTG 152 CGA

(200) The obtained PCR product was separated by agarose gel electrophoresis, then DNA having a target size was extracted from the agarose gel, and an In-Fusion reaction was performed using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.). E. coli JM109 was transformed with the reaction solution, and a target plasmid was extracted from a kanamycin resistant strain. This plasmid was defined as a mutant AtGH3-6 expression plasmid.

(201) (4) A plasmid in which a gene of mutant AtGH3-6 (SEQ ID NOs: 185 and 186) was inserted into NdeI and XhoI sites in a multiple cloning site of pET-28a (+), pET-28a-mutant AtGH3-6 (Mutant No. 36) was purchased from Eurofins Genomics.

Example 2: Construction of Mutant AtGH3-6 Expression Plasmid by Random Mutagenesis

(202) A mutation was introduced into AtGH3-6 using pET-28a-AtGH3-6 (PCT/JP2019/007681) as a template. Mutagenesis was performed using Gene Morph II Random Mutagenesis Kit (Agilent technology) under the following conditions. 1 cycle 95 C., 2 min 30 cycles 95 C., 30 sec 60 C., 30 sec 72 C., 1.2 min 1 cycle 72 C., 10 min 4 C., hold Primers used are as follows.

(203) TABLE-US-00007 TABLE5 Primerforrandommutagenesisusedin preparationofmutantAtGH3-6 Nucleotidesequence(5to3) SEQIDNO TAATACGACTCACTATAGGG 153 ATGCTAGTTATTGCTCAGCGG 154

(204) The obtained DNA fragment of about 2.1 kb was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with a vector-side DNA fragment of pET-28a-AtGH3-6 similarly treated with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, and a plasmid was extracted from a kanamycin resistant strain. This plasmid was defined as a mutant AtGH3-6 expression plasmid library. The obtained plasmid library was introduced into E. coli BL21 (DE3) to obtain a transformant having a mutant AtGH3-6 expression plasmid from a kanamycin resistant strain. A plasmid was extracted from the obtained transformant, and the sequence of AtGH3-6 was confirmed. As a result, the following amino acid substitutions were introduced.

(205) TABLE-US-00008 TABLE 6 Mutation point of mutant AtGH3-6 obtained by random mutagenesis Mutant No. Mutation point R1_3-F11 R350T/G379D R1_5-H9 V311A R3_5-C8 L137I R4_1-A6 Q200E R5_5-D3 K388N R5_5-E7 I123T R6_2-H7 C335S/L390P R6_5-B7 V140I R7_2-C2 N101S R9_3-D4 E483D

Example 3: Purification of Mutant AtGH3-6

(206) The mutant AtGH3-6 expression plasmids were each introduced into E. coli BL21 (DE3) to obtain a transformant having the plasmid from a kanamycin resistant strain. This strain was inoculated into 50 mL of an LB medium containing 50 mg/L kanamycin, and cultured by shaking using a Sakaguchi flask at 37 C. When OD610 reached 0.6, 1 mM IPTG was added, and cultured by shaking at 15 C. for 24 hours. After completion of the culture, bacterial cells were collected from 20 mL of the culture solution by centrifugation and suspended in 4 mL xTractor (trademark) Buffer (Takara Bio Inc.). 8 L of 5 units/L DNase I solution and 40 L of 100 lysozyme solution attached to xTractor (trademark) Buffer Kit (Takara Bio Inc.) were added, and the resulting mixture was stirred by inversion and then left to stand at room temperature for 20 minutes. The supernatant obtained by centrifugation was subjected to TALON (registered trademark) Spin Column (Takara Bio Inc.), and purification was performed according to the manufacturer's protocol. As an equilibration buffer, 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole were used. As an elution buffer, 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 150 mM imidazole were used. The obtained eluate was collected, concentrated using Amicon Ultra-0.5 10 kDa (Merck), and subjected to buffer exchange in 20 mM Tris-HCl (pH 8.0) and 1 mM DTT to obtain a purified enzyme solution. The protein concentration of the obtained purified enzyme solution was measured using a protein assay CBB solution (5-fold concentration) (Nacalai Tesque) and using Quick Start BSA Standard Set (Bio-rad) as a standard sample.

Example 4: Measurement of Activity of Mutant AtGH3-6 to Each Amino Acid Substrate

(207) A 0.2 mL reaction solution having a pH of 8.0, the reaction solution containing 50 mM Tris-HCl, 5 mM L-glutamic acid or L-aspartic acid, 5 mM sodium laurate, 10 mM ATP, 10 mM MgCl.sub.2, 1 mM DTT, and 50 g/mL purified enzyme, was incubated at 25 C. for 24 hours. After completion of the reaction, 0.8 mL of a reaction stop solution (1.4% (w/v) phosphoric acid, 75% (v/v) methanol) was added, and the supernatant after centrifugation was subjected to UPLC-MS analysis. A signal of a molecular weight of N-lauroyl-L-glutamic acid (C12-L-Glu) or N-lauroyl-L-aspartic acid (C12-L-Asp) was extracted by a selected ion recording (SIR) method to confirm a peak area value, and the amount of production was quantified using a calibration curve of a sample.

(208) UPLC-MS analysis conditions are as follows.

(209) Analysis condition 1: Apparatus: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 m 2.150 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile
Gradient: A: B 60:40 to 20:80 Gradient 1.2 min A: B 20:80 to 60:40 Gradient 0.01 min A: B 60:40 Isocratic 0.29 min Flow rate: 0.6 ml/min Injection amount: 2 L Column temperature: 40 C. Ionization method: ESI-negative SIR: C12-L-Glu m/z328.sup. C12-L-Asp m/z314.sup.

(210) Analysis condition 2: Apparatus: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 m 2.1100 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile
Gradient: A: B 60:40 to 20:80 Gradient 2.4 min A: B 20:80 to 60:40 Gradient 0.1 min A: B 60:40 Isocratic 0.5 min Flow rate: 0.6 ml/min Injection amount: 2 L Column temperature: 40 C. Ionization method: ESI-negative SIR: C12-L-Glu m/z328.sup. C12-L-Asp m/z314.sup.

(211) As a result of UPLC-MS analysis, it was confirmed that the production amount of N-lauroyl-L-glutamic acid (C12-L-Glu) and/or a substrate specificity to L-glutamic acid, production amount of C12-L-Glu/production amount of C12-L-Asp, was improved in the mutant AtGH3-6 as compared with the wild type AtGH3-6. WT in Table indicates wild type AtGH3-6.

(212) TABLE-US-00009 TABLE 7-1 N-acylamino acid synthesis activity of each mutant AtGH3-6 to L-glutamic acid and L-aspartic acid as substrate (1) Substrate Product (mM) specificity C12- C12- C12-L-Glu/ No Mutation L-Asp L-Glu C12-L-Asp WT 0.76 0.06 0.08 R18 Y134F 0.85 0.10 0.11 R20 Y134V 0.96 0.08 0.08 R30 S161P 0.75 0.14 0.18 R32 V174A 0.66 0.11 0.16 WT 0.93 0.07 0.08 R55 T336S 1.05 0.09 0.08 R56 M337G 1.13 0.15 0.14 R59 A339G 0.98 0.13 0.14 R61 S340A 0.78 0.10 0.13 WT 1.41 0.09 0.06 R39 V231A 0.98 0.18 0.19 R68 Y344G 1.54 0.11 0.07 R69 Y344A 1.74 0.12 0.07 R70 Y344V 1.35 0.19 0.14 R78 S455T 1.25 0.14 0.11 R1002 T122S/I123T 1.81 0.10 0.05 R1005 R117P/Q533R 0.08 0.14 1.74 WT 1.65 0.09 0.05 R1_3-F11 R350T/G379D 2.16 0.16 0.08 R1_5-H9 V311A 1.82 0.25 0.14 R3_5-C8 L137I 2.84 0.19 0.07 WT 0.55 0.03 0.05 R4_1-A6 Q200E 0.84 0.07 0.09 R5_5-D3 K388N 0.71 0.08 0.11 R5_5-E7 I123T 1.16 0.12 0.10 R7_2-C2 N101S 0.72 0.05 0.07 WT 0.83 0.07 0.09 R6_2-H7 C335S/L390P 0.88 0.11 0.13 R6_5-B7 V140I 1.00 0.10 0.10 R9_3-D4 E483D 1.00 0.09 0.09 WT 1.92 0.18 0.09 4 C576A 1.99 0.38 0.19 WT 2.29 0.15 0.07 7 S161P/V174A 1.67 0.64 0.38 8 S161P/V231A 1.69 0.69 0.41 10 S161P/Y344V 2.52 1.05 0.42 11 V174A/V231A 1.49 0.70 0.47 15 V231A/Y344V 1.63 0.35 0.21 18 R117P/V174A/Q533R 0.23 0.66 2.85 19 R117P/V231A/Q533R 0.38 1.43 3.74 20 R117P/M337G/Q533R 0.13 0.53 4.24 21 R117P/Y344V/Q533R 0.32 0.84 2.65 24 Y344I 2.14 0.17 0.08

(213) TABLE-US-00010 TABLE 7-2 N-acylamino acid synthesis activity of each mutant AtGH3-6 to L-glutamic acid and L-aspartic acid as substrate (2) Substrate Product (mM) specificity C12- C12- C12-L-Glu/ No. Mutation L-Asp L-Glu C12-L-Asp WT 2.37 0.34 0.14 9 S161P/M337G 2.95 2.11 0.72 12 V174A/M337G 2.58 1.62 0.63 13 V174A/Y344V 2.40 0.68 0.28 14 V231A/M337G 1.92 1.12 0.58 17 R117P/S161P/Q533R 0.32 1.07 3.32 WT 2.11 0.23 0.11 16 M337G/Y344V 1.94 0.80 0.41 WT 1.85 0.08 0.04 30 R117P/V174A/V231A/Q533R 0.30 1.26 4.19 32 R117P/V174A/Y344V/Q533R 0.12 0.46 3.76 WT 1.78 0.10 0.06 34 R117P/V231A/Y344V/Q533R 0.08 0.37 4.63 WT 2.28 0.24 0.10 26 R117P/S161P/V174A/M337A/ 0.84 1.79 2.12 Q533R 29 R117P/S161P/Y344V/Q533R 0.59 1.27 2.16 31 R117P/V174A/M337G/ 1.22 2.20 1.81 Q533R 33 R117P/V231A/M337G/ 0.40 1.61 3.98 Q533R WT 2.20 0.26 0.12 27 R117P/S161P/V231A/Q533R 0.26 0.88 3.41 36 R117P/S161P/V174A/V231A/ M337G/ 0.51 1.04 2.03 Y344V/Q533R WT 2.02 0.15 0.08 31 R117P/V174A/M337G/ 0.64 2.14 3.34 Q533R 38 R117P/V174A/V311A/ 0.70 2.47 3.55 M337G/Q533R 39 R117P/L137I/V174A/M337G/ 0.66 2.43 3.66 Q533R 40 R117P/V174A/Q200E/ 0.90 2.88 3.19 M337G/Q533R WT 1.47 0.15 0.10 31 R117P/V174A/M337G/ 0.55 1.72 3.11 Q533R 41 R117P/V174A/M337G/ 0.91 2.26 2.47 K388N/Q533R 46 N101S/R117P/I123T/V174A/ 0.93 2.30 2.46 M337G/Q533R 47 R117P/V174A/M337G/ 0.99 2.49 2.51 Q533R/C576A

(214) TABLE-US-00011 TABLE 7-3 N-acylamino acid synthesis activity of each mutant AtGH3-6 to L-glutamic acid and L-aspartic acid as substrate (3) Substrate Product (mM) specificity C12- C12- C12-L-Glu/ No. Mutation L-Asp L-Glu C12-L-Asp WT 2.02 0.07 0.03 31 R117P/V174A/M337G/ 0.68 1.26 1.86 Q533R 37 R117P/V174A/M337G/ 1.21 1.84 1.51 R350T/G379D/Q533R 42 R117P/I123T/V174A/M337G/ 1.43 2.14 1.50 Q533R 43 N101S/R117P/V174A/M337G/ 0.95 1.72 1.82 Q533R 44 R117P/I123T/V174A/Q200E/ 1.53 2.07 1.35 M337G/Q533R 45 N101S/R117P/V174A/Q200E/ 1.07 1.72 1.60 M337G/Q533R 48 R117P/V174A/Q200E/M337G/ 1.21 2.00 1.65 R350T/G379D/Q533R 49 R117P/I123T/V174A/M337G/ 1.80 2.38 1.32 R350T/G379D/Q533R 50 N101S/R117P/V174A/M337G/ 1.36 2.25 1.66 R350T/G379D/Q533R WT 2.00 0.24 0.12 31 R117P/V174A/M337G/ 0.83 2.01 2.43 Q533R 49 R117P/I123T/V174A/M337G/ 1.73 3.07 1.77 R350T/G379D/Q533R 54 N101S/R117P/I123T/V174A/ 1.41 3.01 2.13 M337G/R350T/G379D/Q533R 55 R117P/I123T/V174A/Q200E/ 1.38 2.83 2.05 M337G/R350T/G379D/Q533R 56 R117P/I123T/V174A/M337G/ 2.23 3.28 1.47 R350T/ G379D/Q533R/C576A 57 R117P/I123T/V174A/M337G/ 1.81 3.00 1.65 R350T/G379D/K388N/Q533R

(215) TABLE-US-00012 TABLE 7-4 N-acylamino acid synthesis activity of each mutant AtGH3-6 to L-glutamic acid and L-aspartic acid as substrate (4) Substrate Product (mM) specificity C12- C12- C12-L-Glu/ No. Mutation L-Asp L-Glu C12-L-Asp WT 1.66 0.10 0.06 31 R117P/V174A/M337G/ 0.81 1.60 1.98 Q533R 49 R117P/I123T/V174A/M337G/ 1.89 2.61 1.38 R350T/G379D/Q533R 56 R117P/I123T/V174A/M337G/ 2.53 3.24 1.28 R350T/G379D/Q533R/C576A 58 N101S/R117P/I123T/V174A/ 2.12 2.92 1.38 M337G/R350T/G379D/ Q533R/C576A 59 R117P/I123T/V174A/Q200E/ 2.31 3.05 1.32 M337G/R350T/G379D/ Q533R/C576A 60 R117P/I123T/V174A/M337G/ 2.38 3.26 1.37 R350T/G379D/K388N/ Q533R/C576A 61 N101S/R117P/I123T/V174A/ 2.26 3.12 1.38 Q200E/M337G/R350T/ G379D/Q533R/C576A 62 N101S/R117P/I123T/V174A/ 2.48 3.11 1.25 M337G/R350T/G379D/ K388N/Q533R/C576A 63 R117P/I123T/V174A/Q200E/ 2.44 3.23 1.33 M337G/R350T/G379D/ K388N/Q533R/C576A 64 N101S/R117P/I123T/V174A/ 2.77 3.25 1.17 Q200E/M337G/R350T/ G379D/K388N/ Q533R/C576A

Example 5: Measurement of Activity of Mutant AtGH3-6 to Each Fatty Acid Substrate

(216) A 0.2 mL reaction solution having a pH of 8.0, the reaction solution containing 50 mM Tris-HCl, 5 mM L-glutamic acid, 5 mM fatty acid sodium (or fatty acid), 10 mM ATP, 10 mM MgCl.sub.2, 1 mM DTT, and 50 g/mL purified enzyme, was incubated at 25 C. for 24 hours. The sodium fatty acid (or fatty acid) to be added to the enzymatic reaction was dissolved in 20% (v/v) Triton X-100 to prepare a 25 mM solution, and the solution was added to the reaction solution so as to have a final concentration of 5 mM. After completion of the reaction, 0.8 mL of a reaction stop solution (1.4% (w/v) phosphoric acid, 75% (v/v) methanol) was added, and the supernatant after centrifugation was subjected to UPLC-MS analysis. A signal of the molecular weight of an assumed N-acyl-L-glutamic acid was extracted by an SIR method, a peak area value was confirmed, and the amount of production was quantified using a calibration curve of a sample. For quantification of C6-L-Glu and C8-L-Glu, C8-L-Glu was used, for quantification of C10-L-Glu, C10-L-Glu was used, and for quantification of N-acyl-L-Glu having an acyl group having a carbon chain length of C12 or more, C12-L-Glu was used as a standard sample. As the sodium fatty acid (or fatty acid), 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 was used. A saturated fatty acid having x carbon atoms is represented by Cx, and an unsaturated fatty acid having x carbon atoms and y carbon-carbon double bonds is represented by Cx: y.

(217) UPLC-MS analysis conditions are as follows: Apparatus: ACQUITY UPLC (Waters) Column: ACQUITY UPLC BEH C18 1.7 m 2.150 mm Column (Waters) Mobile phase A: 0.1% formic acid Mobile phase B: acetonitrile
Gradient: A: B 90:10 to 0:100 Gradient 1.8 min A: B 0:100 Isocratic 0.6 min A: B 0:100 to 90:10 Gradient 0.1 min A: B 90:10 Isocratic 0.5 min Flow rate: 0.6 ml/min Injection amount: 2 L Column temperature: 40 C. Ionization method: ESI-negative

(218) TABLE-US-00013 TABLE 8 MS ions detected by SIR method N-acyl-L-Glu m/z .sup. C6-L-Glu 244 C8-L-Glu 272 C10-L-Glu 300 C12-L-Glu 328 C12:1-L-Glu 326 C14-L-Glu 356 C14:1-L-Glu 354 C16-L-Glu 384 C16:1-L-Glu 382

(219) As a result of UPLC-MS analysis, it was confirmed that the production amounts of various N-acyl-L-glutamic acids each having an acyl group having a carbon chain number of 8 or more were improved in the mutant AtGH3-6 as compared with the wild type AtGH3-6. WT in Table indicates wild type AtGH3-6.

(220) TABLE-US-00014 TABLE 9 N-acylamino acid synthesis activity of each mutant AtGH3-6 to each fatty acid substrate Mutant N-acyl-L-Glu (mM) No. Mutation point C6 C8 C10 C12 C12:1 C14 C14:1 C16 C16:1 WT 1.14 0.30 0.35 0.50 1.03 0.64 0.17 0.07 2.21 31 R117P/V174A/ 0.14 1.37 2.72 3.11 3.12 1.67 1.70 0.24 4.38 M337G/Q533R 49 R117P/I123T/V174A/ 0.22 1.47 3.12 3.12 3.61 1.52 1.95 0.12 4.01 M337G/R350T/ G379D/Q533R 56 R117P/I123T/V174A/ 0.35 1.50 2.89 3.17 3.25 2.62 2.14 0.28 4.97 M337G/R350T/G379D/ Q533R/C576A 61 N101S/R117P/I123T/ 0.41 1.62 2.44 3.80 3.25 2.50 1.92 0.33 3.97 V174A/Q200E/M337G/ R350T/G379D/Q533R/ C576A

Example 6: Construction of Fatty Acid Producing Strain (E. coli)

(221) As a strain that produces a fatty acid from a carbon source such as glucose, an acyl-CoA synthetase (fadD)-deleted/acyl-ACP thioesterase-enhanced E. coli strain (E. coli AfadD/pMW118-Ptac-UcTEopt) was constructed according to the following procedure.

(1) Synthesis of UcTE (acyl-ACP thioesterase derived from Umbellularia californica) gene

(222) Acyl-ACP thioesterase is known as a plant-derived enzyme involved in synthesis of a medium-chain fatty acid. This enzyme can be used to modify a chain length in bacterial fatty acid synthesis. Medium-chain acyl-ACP thioesterase (UcTE, GenBank: M94159) derived from California bay (Umbellularia californica) that predominantly produces lauric acid in oilseeds has been studied and has been used for lauric acid production in E. coli (Voelker and Davies, J. of Bacteriol. 1994; 176 (23): 7320-7327). Although it has been clarified that a transport peptide exists in an N-terminal region of UcTE (SEQ ID NO: 187), it has been indicated that the transport peptide is not essential for an enzymatic activity of UcTE (Feng et al. ACS Chem Biol. 2017; 12 (11): 2830-2836). Therefore, for construction of an expression plasmid of a UcTE gene, a truncated gene containing no transport peptide, that is, a gene encoding a protein having an amino acid sequence in which an initiating methionine is added to amino acid residues at positions 84 to 382 in the amino acid sequence of SEQ ID NO: 3, was used. The sequence of the truncated UcTE gene in which a codon is optimized for expression in E. coli is shown in SEQ ID NO: 4. For the UcTE gene in which a codon is optimized for expression in E. coli, gene synthesis was requested to Eurofins Genomics, and a plasmid in which a DNA fragment containing the gene was inserted into a pEX-K4J1 vector (pEX-K4J1-UcTEopt) was purchased.

(2) Construction of pMW118-Sce-Km plasmid

(223) In order to obtain plasmids available in both E. coli and P. ananatis, an expression vector pMW118-Sce-Km was constructed. This plasmid contains a kan gene (kanamycin resistant marker) and a bla gene (ampicillin resistant marker). Based on a pMW118-placUV5-lacI plasmid (Skorokhodova et al., Biotechnologiya (Russian). 2004; 5:3-21), the pMW118-Sce-Km plasmid (FIG. 1) was constructed. A kan gene (kanamycin resistant marker) encoding aminoglycoside phosphotransferase and a DNA fragment containing an RBS thereof were PCR-amplified using pUC-4K (GenBank/EMBL accession number X06404, Pharmacia) as a template (Mashko et al., Biotekhnologiya. 2001; 5:3-20). Primers used are as follows.

(224) TABLE-US-00015 TABLE10 PrimerforamplificationofDNAfragment containingkangeneandRBSthereof SEQ ID PrimerNo. Nucleotidesequence(5to3) NO P1 TCTGATCTAGATAGGGATAACAGGGTAAT 155 CAACCAATTAACCAATTCTGATTAGAA P2 GGAAAGGATCCGCGGCCGCCACGTTGTGT 156 CTCAAAATCTC

(225) The obtained DNA fragment was inserted into BamHI and XbaI sites of the pMW118-placUV5-lacI plasmid by a ligation reaction with T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation reaction solution and grown on an LB agar medium containing 50 mg/L kanamycin, and then pMW118-Sce-Km was extracted from a kanamycin resistant strain. The structure of the plasmid was confirmed by sequence analysis (FIG. 1).

(3) Construction of pMW118-Ptac-UcTEopt Plasmid

(226) A DNA fragment containing a truncated UcTEopt gene which was fused to RBS (RBST7) derived from a T7 phage and to which a restriction enzyme site was added was PCR-amplified using a pEX-K4J1-UcTEopt plasmid as a template. Primers used are as follows.

(227) TABLE-US-00016 TABLE11 PrimerforamplificationofDNAfragment containingRBST7andtruncatedUcTEoptgene SEQ Primer ID No. Nucleotidesequence(5to3) NO P3 TTTTTTCTAGAAATAATTTTGTTTAACTTTAAGAAG 157 GAGATATACCATGCTAGAGTGGAAACCGAAACCG P4 TTTTTGGATCCTCGAGTTAAACGCGAGGTTCCGCAG 158

(228) A DNA fragment to which a restriction enzyme site was added and which contained a Ptac promoter was PCR-amplified using a chromosomal DNA (Katashkina et al. Molecular Biology. 2005; 39 (5): 719-726) into which a Ptac promoter was introduced as a template. Primers used are as follows.

(229) TABLE-US-00017 TABLE12 PrimerforamplificationofDNAfragment containingPtacpromoter SEQ Primer ID No. Nucleotidesequence(5to3) NO P5 TTTTTAGATCTCCCTGTTGACAATTAATCATCGG 159 P6 CTGTTTCTAGATCCTGTGTGAAATTGTTATCCGC 160

(230) The obtained DNA fragment containing RBST7 and a truncated UcTEopt gene and the obtained DNA fragment containing a Ptac promoter were treated with XbaI, and both DNA fragments were ligated using T4 DNA ligase (Thermo Fisher Scientific). A DNA fragment containing Ptac-RBST7-UcTEopt (SEQ ID NO: 6) to which BglII and an EcoRI sites were added was PCR-amplified using this ligation reaction solution as a template. Primers used are as follows.

(231) TABLE-US-00018 TABLE13 PrimerforamplificationofDNAfragment containingPtac-RBST7-UcTEopt SEQ ID PrimerNo. Nucleotidesequence(5to3) NO P5 TTTTTAGATCTCCCTGTTGACAATTAATCATCGG 161 P7 TTTTTGAATTCGAGCTCGGTACCAATAATTTTGT 162 TTAACTTTAAGAAGGAGATATACC

(232) The obtained DNA fragment was inserted into BglII and EcoRI sites of pMW118-Sce-Km by a ligation reaction with T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation reaction solution and grown on an LB agar medium containing 50 mg/L kanamycin, and then a pMW118-Ptac-UcTEopt plasmid was extracted from a kanamycin resistant strain. The structure of the plasmid was confirmed by sequence analysis (FIG. 2).

(4) Construction of E. coli AfadD/pMW118-Ptac-UcTEopt (Fatty Acid Producing Strain)

(233) For construction of a fatty acid producing strain of E. coli, E. coli K-12 MG1655 (F-lambda-ilvG-rfb-50 rph-ATCC 47076) was used as a base strain. In order to block a fatty acid degradation pathway, in-frame deletion of a fadD gene was performed. Using a PCR-amplified DNA fragment containing attL-kan-attR to which a 40 bp region homologous to the fadD gene was added, deletion by a -Red method was performed in a similar manner to a previously reported method (Katashkina et al., BMC Mol Biol. 2009; 10:34). After electroporation, a strain was grown on an LB agar medium with 50 mg/L kanamycin. The DNA fragment used to replace the fadD gene with attL-kan-attR was PCR-amplified using genome DNA into which a attL-kan-attR cassette was introduced (Katashkina et al., BMC Mol Biol. 2009; 10:34) as a template. Primers used are as follows.

(234) TABLE-US-00019 TABLE14 PrimerforamplificationofDNAfragment containingattL-kan-attR(DNA fragmentforfadDgenedeletion) SEQ ID PrimerNo. Nucleotidesequence(5to3) NO P9 TGAGCTGACGGCGGCAAAAAGTCACCAGTGACTCTTCG 163 GTTGAAGCCTGCTTTTTTATACTAAGTTGG P10 CAACCTGCGTTTGTGAATATGGGGGAGGTAATGACCTT 164 CCCGCTCAAGTTAGTATAAAAAAGCTGAAC

(235) The obtained E. coli MG1655 AfadD:: attL-kan-attR was confirmed by PCR. Primers used are as follows.

(236) TABLE-US-00020 TABLE15 PrimerforconfirmingdeletionoffadDgene PrimerNo. Nucleotidesequence(5to3) SEQIDNO P11 TGCGATGACGACGAACACGC 165 P12 GATTAACCGGCGTCTGACGAC 166

(237) From the obtained strain, a kanamycin resistant marker (kan gene) was removed by a previously reported phage Int/Xis-dependent method (Katashkina et al., BMC Mol Biol. 2009; 10:34). Removal of the marker from a chromosome was confirmed by PCR. Primers used are as follows.

(238) TABLE-US-00021 TABLE16 Primerforconfirmingremovalofkanamycin resistantmarker(kangene) PrimerNo. Nucleotidesequence(5to3) SEQIDNO P11 TGCGATGACGACGAACACGC 167 P12 GATTAACCGGCGTCTGACGAC 168

(239) The obtained strain (E. coli MG1655 fadD::attB) was transformed with a pMW118-Ptac-UcTEopt plasmid by an electroporation method to obtain E. coli fadD/pMW118-Ptac-UcTEopt as a fatty acid producing strain.

Example 7: Construction of Fatty Acid Producing Strain (P. ananatis)

(240) As a strain that produces a fatty acid from a carbon source such as glucose, a fadD-deleted/gcd-deleted/acyl-ACP thioesterase-enhanced P. ananatis strain (P. ananatis SC17 (0) fadD gcd/pMW118-PlacUV5-lacI-UcTEopt) was constructed according to the following procedure.

(1) Construction of pMW118-PlacUV5-lacI-UcTEopt Plasmid

(241) A DNA fragment containing RBST7 and a truncated UcTEopt gene was PCR-amplified using the pMW118-Ptac-UcTEopt plasmid described in Example 6 as a template. Primers used are as follows.

(242) TABLE-US-00022 TABLE17 PrimerforamplificationofDNAfragment containingRBST7andtruncatedUcTEoptgene Primer SEQ No. Nucleotidesequence(5to3) IDNO P4 TTTTTGGATCCTCGAGTTAAACGCGAGGTTCCGCAG 169 P8 TTTTTGAGCTCGAATTCTTAAACGCGAGGTTCCGCAG 170

(243) The obtained DNA fragment was inserted into EcoRI and BamHI sites of a pMW118-Sce-Km plasmid by a ligation reaction with T4 DNA ligase (Thermo Fisher Scientific). E. coli TG1 was transformed with this ligation reaction solution and grown on an LB agar medium containing 50 mg/L kanamycin, and then a pMW118-PlacUV5-lacI-UcTEopt plasmid was extracted from a kanamycin resistant strain. The structure of the plasmid was confirmed by sequence analysis (FIG. 3).

(2) Construction of P. ananatis SC17 (0) fadD gcd/pMW118-PlacUV5-lacI-UcTEopt

(244) For construction of a fatty acid producing strain of P. ananatis, P. ananatis SC17 (0) (Katashkina J I et al., BMC Mol Biol. 2009; 10:34) was used as a base strain. In order to block a fatty acid degradation pathway, in-frame deletion of a fadD gene (PAJ_1453) was performed. Using a PCR-amplified DNA fragment containing attL-kan-attR to which a 40 bp region homologous to the fadD gene was added, deletion by a -Red method was performed in a similar manner to a previously reported method (Katashkina et al., BMC Mol Biol. 2009; 10:34). After electroporation, a strain was grown on an LB agar medium with 50 mg/L kanamycin. The DNA fragment used to replace the fadD gene with attL-kan-attR was PCR-amplified using genome DNA into which a attL-kan-attR cassette was introduced (Katashkina et al., BMC Mol Biol. 2009; 10:34) as a template. Primers used are as follows.

(245) TABLE-US-00023 TABLE18 PrimerforamplificationofDNAfragment containingattL-kan-attR(DNAfragmentfor fadDgenedeletion) Primer SEQ No. Nucleotidesequence(5to3) IDNO P13 CTGACGACGACAGTGATCGAGCAGCTCTTCTTTGGTC 171 AGCTGAAGCCTGCTTTTTTATACTAAGTTGG P14 CTCTGGTTGATCTGTTTGAGCAGGCCGTTTCCCGTTAC 172 GCCGCTCAAGTTAGTATAAAAAAGCTGAAC

(246) The obtained P. ananatis SC17 (0) fadD::attL-kan-attR was confirmed by PCR. Primers used are as follows.

(247) TABLE-US-00024 TABLE19 PrimerforconfirmingdeletionoffadDgene PrimerNo. Nucleotidesequence(5to3) SEQIDNO P15 CAAATGCGATTGTCGAGGCG 173 P16 CCGGCGTTCAACCTGAATCC 174

(248) From the obtained strain, a kanamycin resistant marker (kan gene) was removed by a previously reported phage Int/Xis-dependent method (Katashkina et al., BMC Mol Biol. 2009; 10:34). Removal of the marker from a chromosome was confirmed by PCR. Primers used are as follows.

(249) TABLE-US-00025 TABLE20 Primerforconfirmingremovalofkanamycin resistantmarker(kangene) PrimerNo. Nucleotidesequence(5to3) SEQIDNO P15 CAAATGCGATTGTCGAGGCG 175 P16 CCGGCGTTCAACCTGAATCC 176

(250) The obtained strain was named as P. ananatis SC17 (0) fadD::attB.

(251) Next, deletion of a god gene (PAJ_3473) was performed. Genome DNA (Katashkina et al., Biotekhnologiya. 2019; 35 (2): 3-15) isolated from P. ananatis SC17 (0) gcd::attR-attL80-kan-attR80 using Wizard genomic DNA Purification Kit (Promega) was electroporated into the P. ananatis SC17 (0) fadD::attB strain according to a previously reported method (Katashkina et al., BMC Mol Biol. 2009; 10:34). The introduction of a gcd::attR-attL80-kan-attR80 cassette was confirmed using the following primers.

(252) TABLE-US-00026 TABLE21 Primerforconfirmingdeletionofgcdgene Primer SEQ No. Nucleotidesequence(5to3) IDNO P17 AGGGCAGATAGCAGGCATAA 177 P18 TCTGATTGTTTTCCTGAGTTTTC 178

(253) The obtained strain was named as P. ananatis SC17 (0) fadD::attB gcd::attR-attL80-kan-attR80. From this strain, a kanamycin resistant marker (kan gene) was removed by a previously reported method using a pAH129-cat helper plasmid (Andreeva et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). Removal of the marker was confirmed by PCR. Primers used are as follows.

(254) TABLE-US-00027 TABLE22 Primerforconfirmingremovalofkanamycin resistantmarker(kangene) PrimerNo. Nucleotidesequence(5to3) SEQIDNO P17 AGGGCAGATAGCAGGCATAA 179 P18 TCTGATTGTTTTCCTGAGTTTTC 180

(255) The obtained strain (P. ananatis SC17 (0) fadD::attB gcd::attR-attB80) was transformed with a pMW118-PlacUV5-lacI-UcTEopt plasmid by an electroporation method and grown on an LB agar medium containing 50 to 200 mg/L kanamycin to obtain P. ananatis SC17 (0) fadD gcd/pMW118-PlacUV5-lacI-UcTEopt as a fatty acid producing strain.

Example 8: Synthesis of Lauric Acid from Glucose Using Fatty Acid Producing Strain

(256) Each of the fatty acid producing strains constructed in Examples 6 and 7 was inoculated into a K-medium agar medium (K-medium: LB medium to which 0.5 x M9 salt and 5 g/L D-glucose were added) containing 50 mg/L kanamycin and incubated at 30 C. for 16 hours. The strains evaluated are as follows.

(257) TABLE-US-00028 TABLE 23 Fatty acid producing strain used for evaluating synthesis of lauric acid (fatty acid producing strains constructed in Examples 6 and 7) Strain No. Host Plasmid 1 E. coli fadD pMW118-Ptac-UcTEopt 2 P. ananatis pMW118-PlacUV5- fadDgcd lacI-UcTEopt

(258) The obtained bacterial cells were inoculated into 50 mL of an evaluation medium, and cultured by shaking using 500 mL of Ultra Yield (trademark) Flask (THOMSON) at 30 C. and 220 rpm. When Strain No. 2 was cultured, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as follows.

(259) TABLE-US-00029 TABLE 24 Composition of evaluation medium of fatty acid producing strain Raw material name Concentration D-glucose 40 g/L MgSO.sub.47H.sub.2O 1 g/L (NH.sub.4).sub.2SO.sub.4 16 g/L KH.sub.2PO.sub.4 0.3 g/L KCl 1 g/L MES 10 g/L D-pantothenic acid calcium salt 10 mg/L betaine 1 g/L Bacto Tryptone 1 g/L Bacto Yeast Extract 1 g/L FeSO.sub.47H.sub.2O 10 mg/L MnSO.sub.45H.sub.2O 10 mg/L L-LysHCl 125 mg/L L-Met 100 mg/L 2,6-diaminopimelic acid (DAP) 100 mg/L CaCO.sub.3 30 g/L kanamycin monosulfate 50 mg/L

(260) After completion of the culture, 1 mL of the culture solution was well mixed and centrifuged to obtain bacterial cells. To the 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, and the resulting mixture was mixed with a vortex mixer for three minutes. The supernatant obtained by the centrifugation was subjected to GC and GC-MS analysis.

(261) GC analysis conditions are as follows. Apparatus: GC-2010 (SHIMADZU) Column: DB-FFAP 30 m, ID 0.25 mm, film 0.25 m (Agilent Technologies) Injection amount: 1 L Injection method: Split 50:1 Inlet temperature: 280 C. Column oven: 190 C. (5 min)8 C./min250 C. (7.5 min) Carrier gas: He, linear velocity, 35 cm/sec Detector: FID, 300 C.

(262) GC-MS analysis conditions are as follows. Apparatus: Network GC System (6890N, Agilent Technologies) Mass Selective Detector (5973, Agilent Technologies) Column: DB-FFAP 30 m, ID 0.25 mm, film 0.25 m (Agilent Technologies) Injection amount: 1 L Injection method: Split 50:1 Inlet temperature: 280 C. Column oven: 190 C. (5 min)8 C./min250 C. (7.5 min) Carrier gas: He, linear velocity, 39 cm/see MS temperature: 230 C. (ion source), 150 C. (quadrupole) Scan range: m/z 30-350

(263) The amounts of various fatty acids were quantified from area values of peaks obtained by GC analysis. A sample was prepared by dissolving various fatty acids in 1.4% (w/v) phosphoric acid, 75% (v/v) methanol (containing 1000 ppm tridecylic acid as an internal standard). Correction between samples was performed by comparison with a peak area value of tridecylic acid as an internal standard, and the amounts of various fatty acids per culture solution were calculated. As a result of the analysis, production of various fatty acids containing lauric acid as a main component was confirmed in Strain Nos. 1 and 2. The produced various fatty acids were identified by retention time in GC analysis and GC-MS analysis.

(264) TABLE-US-00030 TABLE 25 Production amounts of various fatty acids produced by fatty acid producing strain Fatty acid (g/L) Strain No. Strain sp. C12 C12:1 C14 C14:1 C16 C16:1 C18:1 1 E. coli 1.5 0.1 0.2 0.0 0.2 0.0 0.2 0.0 ND ND ND 2 P. ananatis 1.2 0.2 0.2 0.0 0.2 0.0 0.3 0.0 0.1 0.0 0.1 0.0 0.2 0.0 ND: not detected

Example 9: Construction of N-Acylamino Acid Producing Strain

(265) As a strain that produces an N-acylamino acid from a carbon source such as glucose, a strain in which a wild type AtGH3-6 or a mutant AtGH3-6 expression unit was introduced into each of the fatty acid producing strains constructed in Examples 6 and 7 was constructed according to the following procedure.

(266) For a promoter PphoC_SDatc sequence (SEQ ID NO: 5), DNA synthesis was requested to Thermo Fisher Scientific, and a plasmid in which a DNA fragment containing the sequence was inserted into EcoRI and NdeI sites was purchased. A DNA fragment containing PphoC_SDatc was PCR-amplified using this plasmid as a template. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) under the following conditions. 1 cycle 98 C., 30 sec 30 cycles 98 C., 10 sec 60 C., 15 sec 72 C., 5 sec 1 cycle 72 C., 5 min 4 C., hold Primers used are as follows.

(267) TABLE-US-00031 TABLE26 PrimerforamplificationofDNAfragment containingPphoC_SDatc Nucleotidesequence(5to3) SEQIDNO CCATGATTACGAATTCATTTTTTCAATGTG 181 ATGGATTCCTCCTTACGGTGTTATATGTCC 182

(268) A DNA fragment containing a wild type or mutant AtGH3-6 gene was PCR-amplified 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 constructed in Example 1 (ID indicates Mutant No.) as a template. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) under the following conditions. 1 cycle 98 C., 30 sec 30 cycles 98 C., 10 sec 60 C., 15 sec 72 C., 10 sec 1 cycle 72 C., 5 min 4 C., hold Primers used are as follows.

(269) TABLE-US-00032 TABLE27 PrimersforamplificationofDNAfragment containingwildtypeormutantAtGH3-6gene Nucleotidesequence(5to3) SEQIDNO TAAGGAGGAATCCATATGCCGGAAGCACCA 183 GATCCCCGGGTACCGAGCTCCTCGAGTTAATTACT 184

(270) A PCR product containing PphoC_SDatc, a PCR product containing a wild type or mutant AtGH3-6 gene, and pHSG398 (Takara Bio Inc.) treated with EcoRI and SacI were separated from each other by agarose gel electrophoresis, then a DNA having a target size was extracted from the agarose gel, and an In-Fusion reaction was performed using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.). E. coli JM109 was transformed with the reaction solution, and a target plasmid (plasmid containing PphoC_SDate and AtGH3-6 expression units) was extracted from a chloramphenicol resistant strain. This plasmid was defined as pHSG398-PphoC-AtGH3-6 (SDatc), pHSG398-PphoC-AtGH3-6 ID31 (SDatc), pHSG398-PphoC-AtGH3-6 ID49 (SDatc), pHSG398-PphoC-AtGH3-6 ID56 (SDatc), or pHSG398-PphoC-AtGH3-6 ID61 (SDatc). Each of the fatty acid producing strains constructed in Examples 6 and 7 (E. coli fadD/pMW118-Ptac-UcTEopt and P. ananatis fadD gcd/pMW118-PlacUV5-lacI-UcTEopt) was transformed with the obtained target plasmid and pHSG398 (negative control) to obtain a transformant having the plasmid from kanamycin and chloramphenicol resistant strains. The obtained transformant was defined as an N-acylamino acid producing strain. The strains constructed are as follows.

(271) TABLE-US-00033 TABLE 28 N-acylamino acid producing strain Strain No. Host Plasmid (1) Plasmid (2) 3 E. coli pMW118- pHSG398 4 fadD Ptac- pHSG398-PphoC-AtGH3-6 UcTEopt (SDatc) 5 pHSG398-PphoC-AtGH3-6 ID31 (SDatc) 6 pHSG398-PphoC-AtGH3-6 ID49 (SDatc) 7 pHSG398-PphoC-AtGH3-6 ID56 (SDatc) 8 pHSG398-PphoC-AtGH3-6 ID61 (SDatc) 9 P. pMW118- pHSG398 10 ananatis PlacUV5- pHSG398-PphoC-AtGH3-6 fadD lacI- (SDatc) 11 ged UcTEopt pHSG398-PphoC-AtGH3-6 ID31 (SDatc) 12 pHSG398-PphoC-AtGH3-6 ID49 (SDatc) 13 pHSG398-PphoC-AtGH3-6 ID56 (SDatc) 14 pHSG398-PphoC-AtGH3-6 ID61 (SDatc)

Example 10: Synthesis of N-Lauroyl-L-Glutamic Acid from Glucose Using N-Acylamino Acid Producing Strain

(272) The N-acylamino acid producing strain constructed in Example 9 was inoculated into an LB agar medium containing 50 mg/L kanamycin and 25 mg/L chloramphenicol and incubated at 30 C. for 16 hours. The obtained bacterial cells were inoculated into 3 mL of an evaluation medium, and cultured by shaking using a test tube at 30 C. and 120 rpm for 48 hours. When Strain Nos. 9 to 14 were cultured, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as follows.

(273) TABLE-US-00034 TABLE 29 Composition of evaluation medium of N-acylamino acid producing strain Raw material name Concentration D-glucose 40 g/L MgSO.sub.47H.sub.2O 1 g/L (NH.sub.4).sub.2SO.sub.4 16 g/L KH.sub.2PO.sub.4 0.3 g/L KCl 1 g/L MES 10 g/L D-pantothenic acid calcium salt 10 mg/L betaine 1 g/L Bacto Tryptone 1 g/L Bacto Yeast Extract 1 g/L FeSO.sub.47H.sub.2O 10 mg/L MnSO.sub.45H.sub.2O 10 mg/L L-LysHCl 125 mg/L L-Met 100 mg/L 2,6-diaminopimelic acid (DAP) 100 mg/L CaCO.sub.3 30 g/L kanamycin monosulfate 50 mg/L chloroamphenicol 25 mg/L

(274) After completion of the culture, the culture solution was well mixed. To 100 L of the culture solution, 900 L of 1.4% (w/v) phosphoric acid, 75% (v/v) methanol was added, and the resulting mixture was mixed with a vortex mixer for three minutes. The supernatant obtained by centrifugation was appropriately diluted with 1.4% (w/v) phosphoric acid, 75% (v/v) methanol, and subjected to UPLC-MS analysis. UPLC-MS analysis conditions are as described in Example 5.

(275) As a result of UPLC-MS analysis, when the negative control (pHSG398) and the wild type AtGH3-6 were expressed, various N-acyl-L-glutamic acids were hardly produced, but when the mutant AtGH3-6 (IDs 31, 49, 56, and 61) was expressed, it was confirmed that various N-acyl-L-glutamic acids were produced. In Table, Control indicates the negative control and WT indicates the wild type AtGH3-6.

(276) TABLE-US-00035 TABLE 30 Production amounts of various N-acyl-L-glutamic acids produced by each N- acylamino acid producing strain Strain No. N-acyl-L-Glu (g/L) Strain C10 C12 C12:1 C14 C14:1 C16 C16:1 3 E. coli Control 0.0000 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 4 WT 0.0000 0.0001 0.0001 0.0000 0.0000 0.0000 0.0001 5 ID31 0.0012 0.1296 0.0682 0.0003 0.0053 0.0001 0.0003 6 ID49 0.0019 0.1474 0.0863 0.0009 0.0086 0.0003 0.0012 7 ID56 0.0036 0.1942 0.1188 0.0017 0.0211 0.0006 0.0018 8 ID61 0.0028 0.1745 0.1059 0.0012 0.0149 0.0003 0.0014 9 P. ananatis Control 0.0000 0.0022 0.0000 0.0000 0.0000 0.0000 0.0000 10 WT 0.0011 0.0345 0.0183 0.0007 0.0008 0.0000 0.0003 11 ID31 0.0073 0.7553 0.1953 0.0104 0.1003 0.0001 0.0016 12 ID49 0.0075 0.7029 0.2248 0.0341 0.1200 0.0008 0.0132 13 ID56 0.0069 0.7486 0.1905 0.0125 0.0892 0.0004 0.0054 14 ID61 0.0042 0.7613 0.1522 0.0143 0.0806 0.0004 0.0069

Example 11: Synthesis of N-Lauroyl-L-Aspartic Acid from Glucose Using N-Acylamino Acid Producing Strain

(277) The N-acylamino acid producing strain constructed in Example 9 was inoculated into an LB agar medium containing 50 mg/L kanamycin and 25 mg/L chloramphenicol and incubated at 30 C. for 16 hours. The obtained bacterial cells were inoculated into 3 mL of an evaluation medium, and cultured by shaking using a test tube at 30 C. and 120 rpm for 48 hours. When Strain Nos. 9 to 14 were cultured, 1 mM IPTG was added to the medium. For the composition of the evaluation medium, 10 g/L L-aspartic acid was added to the composition described in Example 10.

(278) After completion of the culture, the culture solution was well mixed. To 100 L of the culture solution, 900 L of 1.4% (w/v) phosphoric acid, 75% (v/v) methanol was added, and the resulting mixture was mixed with a vortex mixer for three minutes. The supernatant obtained by centrifugation was appropriately diluted 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. For quantification of C10-L-Asp, C10-L-Asp was used as a standard sample, and for quantification of N-acyl-L-Asp having an acyl group having a carbon chain length of C12 or more, C12-L-Asp was used as a standard sample.

(279) TABLE-US-00036 TABLE 31 MS ion detected by SIR method N-acyl-L-Asp m/z .sup. C10-L-Asp 286 C12-L-Asp 314 C12:1-L-Asp 312 C14-L-Asp 342 C14:1-L-Asp 340 C16-L-Asp 370 C16:1-L-Asp 368

(280) As a result of UPLC-MS analysis, in the negative control (pHSG398), various N-acyl-L-aspartic acids were hardly produced, but when the wild type AtGH3-6 or the mutant AtGH3-6 (IDs 31, 49, 56, and 61) was expressed, it was confirmed that various N-acyl-L-aspartic acid acids were produced. In Table, Control indicates the negative control and WT indicates the wild type AtGH3-6.

(281) TABLE-US-00037 TABLE 32 Production amounts of various N-acyl-L-aspartic acids produced by each N- acylamino acid producing strain Strain N-acyl-L-Asp (g/L) No. Strain C10 C12 C12:1 C14 C14:1 C16 C16:1 3 E. coli Control 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 4 WT 0.0000 0.0003 0.0001 0.0001 0.0002 0.0001 0.0008 5 ID31 0.0000 0.0012 0.0005 0.0000 0.0001 0.0000 0.0001 6 ID49 0.0001 0.0025 0.0012 0.0001 0.0003 0.0000 0.0001 7 ID56 0.0001 0.0007 0.0017 0.0002 0.0006 0.0000 0.0002 8 ID61 0.0000 0.0022 0.0008 0.0001 0.0003 0.0000 0.0001 9 P. ananatis Control 0.0000 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 10 WT 0.0087 0.1560 0.0912 0.0036 0.0566 0.0003 0.0054 11 ID31 0.0002 0.0816 0.0255 0.0008 0.0065 0.0001 0.0010 12 ID49 0.0006 0.1178 0.0341 0.0028 0.0140 0.0002 0.0042 13 ID56 0.0002 0.0939 0.0277 0.0015 0.0094 0.0001 0.0019 14 ID61 0.0006 0.1359 0.0375 0.0032 0.0171 0.0002 0.0046

Example 12: Evaluation of Foaming of Culture Solution of N-Acylamino Acid Producing Strain

(282) The N-acylamino acid producing strain constructed in Example 9 was inoculated into 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.

(283) TABLE-US-00038 TABLE 33 N-acylamino acid producing strain used for evaluation of foaming of culture solution (N-acylamino acid producing strain constructed in Example 9) Strain No. Host Plasmid (1) Plasmid (2) 3 E. coli pMW118- pHSG398 4 Ptac- pHSG398-PphoC-AtGH3-6 (SDatc) 8 fadD UcTEopt pHSG398-PphoC-AtGH3-6 ID61 (SDatc) 9 P. pMW118- pHSG398 10 ananatis PlacUV5- pHSG398-PphoC-AtGH3-6 (SDatc) 14 fadD lacI- pHSG398-PphoC-AtGH3-6 ID61 gcd UcTEopt (SDatc)

(284) The obtained bacterial cells were inoculated into 50 mL of an evaluation medium, and cultured by shaking using 500 mL of Ultra Yield (trademark) Flask (THOMSON) at 30 C. and 220 rpm. When Strain Nos. 9, 10, and 14 were cultured, 1 mM IPTG was added to the medium. The composition of the evaluation medium is as described in Example 10.

(285) After completion of the culture, the culture solution was well mixed and centrifuged to obtain a culture supernatant. To 100 L of the culture supernatant, 900 L of 1.4% (w/v) phosphoric acid, 75% (v/v) methanol was added, and the resulting mixture was mixed with a vortex mixer for three minutes. The supernatant obtained by centrifugation was appropriately diluted with 1.4% (w/v) phosphoric acid, 75% (v/v) methanol, and subjected to UPLC-MS analysis. UPLC-MS analysis conditions are as described in Example 5.

(286) As a result of UPLC-MS analysis, when the negative control (pHSG398) and the wild type AtGH3-6 were expressed, various N-acyl-L-glutamic acids were hardly produced, but when the mutant AtGH3-6 (ID 61) was expressed, it was confirmed that various N-acyl-L-glutamic acids were produced. In Table, Control indicates the negative control, WT indicates the wild type AtGH3-6, and total indicates the total concentration of N-acyl-L-glutamic acids of C10, C12, C12: 1, C14, C14: 1, C16, and C16: 1 each having an acyl group.

(287) TABLE-US-00039 TABLE 34 Production amounts of various N-acyl-L-glutamic acids produced by each N- acylamino acid producing strain in culture supernatant Strain No. N-acyl-L-Glu (g/L) strain C10 C12 C12:1 C14 C14:1 C16 C16:1 total 3 E. coli Control 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.001 4 WT 0.000 0.001 0.001 0.000 0.000 0.000 0.000 0.002 8 ID61 0.003 0.094 0.094 0.001 0.014 0.000 0.001 0.205 9 P. ananatis Control 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.001 10 WT 0.001 0.009 0.011 0.000 0.002 0.000 0.000 0.024 14 ID61 0.003 0.672 0.177 0.032 0.092 0.001 0.013 0.990

(288) In order to evaluate foaming of the obtained culture supernatants, the pH of 4 mL of each of the culture supernatants was adjusted to 5.0 with HCl, and then ultrapure water was added thereto to make the volume thereof 5 mL. As a control, a solution was prepared in which N-lauroyl-L-glutamic acid (AMISOFT (registered trademark) LA-D) was dissolved in ultrapure water, and the pH thereof was adjusted to 5.0 with NaOH. Furthermore, a solution was prepared in which an N-lauroyl-L-glutamic acid (AMISOFT (registered trademark) LA-D) solution was added to 4 mL of the culture supernatant of the negative control (pHSG398), the pH thereof was adjusted to 5.0 with HCl, and then ultrapure water was added thereto to make the volume thereof 5 mL.

(289) 2 mL of each sample was put in a screw tube (total length 105 mm, mouth inner diameter 10.0 mm, body diameter 16.5 mm) and incubated in a water bath at 35 C. for 10 minutes. Thereafter, the screw tube was held in the hand and shaken up and down for 10 seconds, and the height of foam immediately after shaking was measured.

(290) As a result, stronger foaming was confirmed in a case where the mutant AtGH3-6 (ID 61) was expressed as compared with a case where the negative control (pHSG 398) or the wild type AtGH3-6 was expressed. Therefore, this indicates that the N-acyl-L-glutamic acid produced by the N-acylamino acid producing strain is available as a surfactant application. In Table, Control indicates the negative control, WT indicates the wild type AtGH3-6, and total N-acyl-L-Glu indicates the total concentration of N-acyl-L-glutamic acids of C10, C12, C12: 1, C14, C14: 1, C16, and C16: 1 each having an acyl group.

(291) TABLE-US-00040 TABLE 35 Height of foam after culture supernatant of each N-acylamino acid producing strain is shaken Sample Strain Total N-acyl-L-Glu Height of No. No. Strain (g/L) in sample foam (mm) 1 3 E. coli Control 0.00 1.0 0.0 2 4 WT 0.00 4.0 0.0 3 8 ID61 0.16 16.5 0.5 4 C12-L-Glu solution (control) 0.16 10.5 2.5 5 Sample No. 1 + C12-l-Glu 0.16 5.5 1.5 solution (control) 6 9 P. ananatis Control 0.00 4.5 2.5 7 10 WT 0.02 4.0 2.0 8 14 ID61 0.79 33.0 0.0 9 C12-L-Glu solution (control) 0.79 11.0 1.0 10 Sample No. 6 + C12-l-Glu 0.79 16.0 1.0 solution (control)

INDUSTRIAL APPLICABILITY

(292) The present invention is useful for production of an N-acyl-amino group-containing compound that can be used for a cosmetic material (particularly a surfactant) and the like.

(293) Sequence Listing Free Text

(294) SEQ ID NOs: 1 and 2 represent the amino acid sequence of AtGH3-6 and a nucleotide sequence encoding the amino acid sequence in which a codon is optimized for expression in Escherichia coli, respectively.

(295) SEQ ID NOs: 3 and 4 represent an amino acid sequence of medium-chain acyl-ACP thioesterase (UcTE, GenBank: M94159) derived from California bay (Umbellularia californica) (amino acids 1 to 83 each represent a transport peptide) and a nucleotide sequence encoding an amino acid sequence in which an initiating methionine is added to amino acid residues at positions 84 to 382 in the amino acid sequence (a codon is optimized for expression in Escherichia coli), respectively.

(296) SEQ ID NO: 5 represents a nucleotide sequence of PphoC_SDatc.

(297) SEQ ID NO: 6 represents a nucleotide sequence of Ptac-RBST7-UcTEopt (nucleotides 7 to 71 each represent a Ptac promoter, nucleotides 82 to 110 each represent RBST7, and nucleotides 117 to 1019 each represent a UcTEopt gene).

(298) SEQ ID NOs: 7 to 184 each represent a nucleotide sequence of a primer.

(299) SEQ ID NOs: 185 and 186 represent an amino acid sequence of the mutant AtGH3-6 and a nucleotide sequence encoding the amino acid sequence in which a codon is optimized for expression in Escherichia coli, respectively.

(300) SEQ ID NO: 187 represents an amino acid sequence of a transport peptide of UcTE.