Method of producing N-acyl-amino group-containing compound

Patent metadata

Field	Value
Publication number	US-12281340-B2
Application number	US-202017025173-A
Country	US
Kind code	B2
Filing date	Sep 18, 2020
Priority date	Apr 13, 2018
Publication date	Apr 22, 2025
Grant date	Apr 22, 2025

Abstract

A method efficiently produces a compound containing an N-acyl-amino group by an enzymatic method. Specifically, a method of producing a compound containing an N-acyl-amino group includes producing the compound containing an N-acyl-amino group by reacting a compound containing an amino group with a compound containing a carboxyl group in the presence of an enzyme having an ability to bond a carboxyl group and an amino group in an ATP dependent manner to form an amide bond.

First claim

The invention claimed is:

1. A method of producing a compound containing an N-acyl-amino group, the method comprising:
reacting a compound containing an amino group with a fatty acid in a culture medium comprising a microorganism, wherein the microorganism produces an enzyme, and said reacting results in production of the compound containing an N-acyl-amino group, wherein the fatty acid is a saturated fatty acid having 6 to 18 carbon atoms, and
isolating the compound containing an N-acyl-amino group from the culture medium,
wherein the enzyme has an ability to bond a carboxyl group and an amino group in an ATP dependent manner to form an amide bond, wherein the enzyme is selected from the group consisting of:
(A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9,
(B) a protein comprising an amino acid sequence containing one to twenty amino acid substitutions, deletions, insertions, or additions in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity, and
(C) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity.

2. The method according to claim 1 , wherein the enzyme is selected from the group consisting of:
(A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9;
(B) a protein comprising an amino acid sequence containing one to ten amino acid substitutions, deletions, insertions, or additions in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity; and
(C) a protein comprising an amino acid sequence having 98% or more identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity.

3. The method according to claim 1 , wherein the compound containing an amino group is a compound containing an amino group having an anionic group.

4. The method according to claim 1 , wherein the compound containing an amino group is an amino acid or a peptide.

5. The method according to claim 4 , wherein the compound containing an amino group is an α-amino acid, a β-amino acid, or a γ-amino acid, or a dipeptide thereof.

6. The method according to claim 4 , wherein the amino acid is an L-amino acid or a D-amino acid.

7. The method according to claim 1 , wherein the compound containing an amino group is selected from the group consisting of:
(1) an amino acid selected from the group consisting of:
(a) an α-amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine;
(b) β-alanine;
(c) a γ-aminobutyric acid; and
(d) sarcosine;

(2) taurine; and
(3) a dipeptide selected from the group consisting of aspartylphenylalanine, glycylglycine, and alanylhistidine.

8. The method according to claim 1 , wherein the fatty acid has 6 to 12 carbon atoms.

9. The method according to claim 1 , wherein said microorganism is:
(i) a microorganism comprising a heterologous expression unit containing a polynucleotide encoding said enzyme and a promoter operably linked thereto;
(ii) a microorganism comprising an expression unit containing a polynucleotide encoding said enzyme and a promoter operably linked thereto in a non-natural genomic region or a non-genomic region; or
(iii) a microorganism comprising a polynucleotide encoding said enzyme in multiple copy number in an expression unit.

10. The method according to claim 1 , wherein said microorganism is a bacterium belonging to Enterobacteriaceae.

11. The method according to claim 10 , wherein said bacterium is Escherichia coli.

12. A method of producing a compound containing an N-acyl-amino group, the method comprising:
reacting a compound containing an amino group with a fatty acid in a culture medium comprising a microorganism, wherein the microorganism produces an enzyme, and said reacting results in production of the compound containing an N-acyl-amino group, and
isolating the compound containing an N-acyl-amino group from the culture medium,
wherein the enzyme has an ability to bond a carboxyl group and an amino group in an ATP dependent manner to form an amide bond,

wherein the enzyme is selected from the group consisting of:
(A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9,
(B) a protein comprising an amino acid sequence containing one to twenty amino acid substitutions, deletions, insertions, or additions in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity, and
(C) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 9, and said protein having an N-acylase activity;

wherein the fatty acid is selected from the group consisting of caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonic acid (C9), capric acid (C10), undecylic acid (C11), lauric acid (C12), tridecylic acid (C13), myristic acid (C14), pentadecylic acid (C15), palmitic acid, palmitoleic acid, sapienic acid (C16), margaric acid (C17), stearic acid, α-linolenic acid, γ-linolenic acid, linoleic acid, vaccenic acid, oleic acid (C18), coconut oil fatty acid, palm fatty acid, hardened beef tallow fatty acid, and combinations thereof.

13. The method of claim 1 , wherein the fatty acid is selected from the group consisting of caproic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid, and combinations thereof.

14. The method of claim 1 , wherein the fatty acid is selected from the group consisting of caproic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid, linoleic acid, and combinations thereof.

Assignees

• Ajinomoto Kk

Inventors

• Nagata Hidemi
• Danjou Keiko
• Takakura Jun
• Nozaki Hiroyuki

Classifications

• C12Y602/0103

  Phenylacetate--CoA ligase (6.2.1.30) · CPC title

• C12Y305/01014

  Aminoacylase (3.5.1.14) · CPC title

• C12Y302/01021

  Beta-glucosidase (3.2.1.21) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N9/93

  Ligases (6) · CPC title