Microorganism producing eicosapentaenoic acid and method for producing eicosapentaenoic acid

Patent metadata

Field	Value
Publication number	US-11613728-B2
Application number	US-201917266984-A
Country	US
Kind code	B2
Filing date	Aug 9, 2019
Priority date	Aug 10, 2018
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

An object of the present invention is to provide a microorganism that efficiently produces EPA and a method for producing EPA using the microorganism. The present invention relates to a microorganism having an ability to produce docosahexaenoic acid (DHA), wherein the microorganism contains a protein composed of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 in the amino acid sequence represented by SEQ ID NO: 2 has been substituted with another amino acid residue (mutated OrfB), and is capable of producing eicosapentaenoic acid (EPA), and the like.

First claim

The invention claimed is:

1. A microorganism having an ability to produce docosahexaenoic acid (DHA), wherein the microorganism contains a mutated OrfB protein composed of an amino acid sequence represented by SEQ ID NO: 2 except for at least one amino acid substitution selected from the group consisting of: N6S, F65L, F230L/N/G/D/A, I231T, and D275G, and wherein the mutated OrfB gives the microorganism ability to produce eicosapentaenoic acid (EPA).

2. A microorganism containing a mutated OrfB protein composed of an amino acid sequence represented by SEQ ID NO: 2 except that at least one amino acid at position 6, 65, 230, 231 or 275 has been substituted with another amino acid.

3. The microorganism according to claim 1 , wherein the microorganism having an ability to produce DHA is a Labyrinthulomycetes microorganism.

4. The microorganism according to claim 3 , wherein the Labyrinthulomycetes microorganism is a Labyrinthulomycetes microorganism belonging to the genus Aurantiochytrium , the genus Thraustochytrium , the genus Ulkenia , the genus Parietichytrium , the genus Labyrinthula , the genus Aplanochytrium , the genus Oblongichytrium , or the genus Schizochytrium.

5. The microorganism according to claim 1 , wherein the microorganism having an ability to produce DHA is a microorganism in which genes encoding respective domains described in the following (a) to (j) having an activity of synthesizing DHA have been introduced into a microorganism that does not have a DHA metabolic pathway:
(a) a β-ketoacyl-ACP synthase (hereinafter referred to as KS) domain;
(b) a malonyl-CoA:ACP acyltransferase (hereinafter referred to as MAT) domain;
(c) an acyl carrier protein (ACP) domain;
(d) a ketoreductase (hereinafter referred to as KR) domain;
(e) a polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain;
(f) a chain elongation factor (hereinafter, referred to as CLF) domain;
(g) an acyltransferase (hereinafter referred to as AT) domain;
(h) a FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain;
(i) an enoyl-ACP reductase (hereinafter referred to as ER) domain; and
(j) a phosphopantetheine transferase (hereinafter referred to as PPT) domain.

6. The microorganism according to claim 5 , wherein the microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genus Escherichia , the genus Bacillus , the genus Corynebacterium , the genus Yarrowia , the genus Saccharomyces , the genus Candida , or the genus Pichia.

7. A method for producing EPA or an EPA-containing composition, comprising:
culturing the microorganism according to claim 1 in a culture medium so as to produce and accumulate EPA or an EPA-containing composition in a culture, and
collecting EPA or the EPA-containing composition from the culture.

8. The microorganism according to claim 2 , wherein the microorganism is a Labyrinthulomycetes microorganism.

9. The microorganism according to claim 8 , wherein the Labyrinthulomycetes microorganism is a Labyrinthulomycetes microorganism belonging to the genus Aurantiochytrium , the genus Thraustochytrium , the genus Ulkenia , the genus Parietichytrium , the genus Labyrinthula , the genus Aplanochytrium , the genus Oblongichytrium , or the genus Schizochytrium.

10. The microorganism according to claim 2 , wherein the microorganism does not naturally have a DHA metabolic pathway and has been modified to be able to synthesize DHA by introducing genes encoding domains (a) to (j):
(a) a β-ketoacyl-ACP synthase (hereinafter referred to as KS) domain;
(b) a malonyl-CoA:ACP acyltransferase (hereinafter referred to as MAT) domain;
(c) an acyl carrier protein (ACP) domain;
(d) a ketoreductase (hereinafter referred to as KR) domain;
(e) a polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain;
(f) a chain elongation factor (hereinafter, referred to as CLF) domain;
(g) an acyltransferase (hereinafter referred to as AT) domain;
(h) a FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain;
(i) an enoyl-ACP reductase (hereinafter referred to as ER) domain; and
(j) a phosphopantetheine transferase (hereinafter referred to as PPT) domain.

11. The microorganism according to claim 10 , wherein the microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genus Escherichia , the genus Bacillus , the genus Corynebacterium , the genus Yarrowia , the genus Saccharomyces , the genus Candida , or the genus Pichia.

12. A method comprising culturing the microorganism according to claim 2 in a culture medium.

Assignees

• Kyowa Hakko Bio Co Ltd

Inventors

• Dairi Tohru
• Satoh Yasuharu
• Hayashi Shohei
• NAKA Mai
• Ujihara Tetsuro

Classifications

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12R2001/90

  Protozoa {; Processes using protozoa} · CPC title

• C12P7/64

  Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats · CPC title

• C12N1/105 Primary

  Protozoal isolates · CPC title