IP Library › Granted Patent US 12,241,101
Granted Patent B2
US 12,241,101 · App. 17/266,976 · Granted Mar 4, 2025

Microorganism producing polyunsaturated fatty acid and method for producing polyunsaturated fatty acid

Inventors: Tohru Dairi (Sapporo, JP); Yasuharu Satoh (Sapporo, JP); Shohei Hayashi (Sapporo, JP); Yasushi Ogasawara (Sapporo, JP); Tetsuro Ujihara (Tokyo, JP)
Assignee: KYOWA HAKKO BIO CO., LTD.
C12N9/93C12N9/001C12N9/1029C12N9/1288C12P7/6427C12Y103/01009C12Y203/01038C12Y203/01041C12Y207/08007
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Quick Facts
Patent No.
US 12,241,101
App. No.
17/266,976
Granted
Mar 4, 2025
Kind
B2
Abstract

An object of the present invention is to provide a microorganism that efficiently produces a PUFA and a method for producing a PUFA using the microorganism. The present invention relates to a microorganism capable of producing a polyunsaturated fatty acid (PUFA), in which a gene encoding an exogenous polyketide synthase dehydratase (PS-DH) domain having a higher activity against 3-hydroxyhexanoyl acyl carrier protein (3-hydroxyhexanoyl ACP) than an endogenous FabA-like β-hydroxyacyl-ACP dehydratase (FabA-DH) domain has been introduced into a microorganism having a PUFA metabolic pathway, and the like.

Claims (123)

1. A microorganism capable of producing a ω-6 polyunsaturated fatty acid (PUFA),

in which the reactivity of a polyketide synthase dehydratase (PS-DH) domain with 3-hydroxyhexanoyl acyl carrier protein (3-hydroxyhexanoyl ACP) in the microorganism is higher than the reactivity of a FabA-like β-hydroxyacyl-ACP dehydratase (FabA-DH) domain with 3-hydroxyhexanoyl ACP in the microorganism, and

wherein the microorganism has an anaerobic pathway involving a protein complex having PUFA-PKS activity,

wherein proteins constituting the protein complex having PUFA-PKS activity are one of the following combinations (i) to (v):

(i) PhoA, PhoB, PhoC, PhoD, EpaE, AraB,

(ii) EpaA, PhoB, PhoC, PhoD, EpaE, AraB,

(iii) EpaA, AraB, AraC, AraD, EpaE,

(iv) PhoA, AraB, AraC, AraD, EpaE, and

(v) OrfA, OrfB, OrfC, EpaE, AraB, and

wherein

the amino acid sequence of AraB is SEQ ID NO: 58,

the amino acid sequence of AraC is SEQ ID NO: 60,

the amino acid sequence of AraD is SEQ ID NO: 62,

the amino acid sequence of PhoA is SEQ ID NO: 66,

the amino acid sequence of PhoB is SEQ ID NO: 68,

the amino acid sequence of PhoC is SEQ ID NO: 70,

the amino acid sequence of PhoD is SEQ ID NO: 72,

the amino acid sequence of EpaA is SEQ ID NO: 74,

the amino acid sequence of EpaE is SEQ ID NO: 82,

the amino acid sequence of OrfA is SEQ ID NO: 84,

the amino acid sequence of OrfB is SEQ ID NO: 86 or 90, and

the amino acid sequence of OrfC is SEQ ID NO: 88 or 92.

2. The microorganism according to claim 1 , wherein the microorganism capable of producing a ω-6 PUFA is a microorganism having a ω-3 PUFA metabolic pathway.

3. The microorganism according to claim 1 , wherein

the microorganism capable of producing a ω-6 PUFA is a microorganism having an eicosapentaenoic acid (EPA) metabolic pathway or docosahexaenoic acid (DHA) metabolic pathway, and

the ω-6 PUFA that can be produced is arachidonic acid (ARA) or docosapentaenoic acid (DPA).

4. The microorganism according to claim 1 , wherein the microorganism capable of producing a ω-6 PUFA is derived from the genus Shewanella , the genus Photobacterium , the genus Moritella , the genus Colwellia , 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. A microorganism capable of producing a ω-6 PUFA,

in which genes encoding respective domains described in the following (a) to (j) having an activity of synthesizing a PUFA (hereinafter referred to as PUFA-PKS activity) have been introduced into a microorganism that does not have a PUFA 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 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-DH domain;

(i) an enoyl-ACP reductase (hereinafter referred to as ER) domain; and

(j) a phosphopantetheine transferase (hereinafter referred to as PPT) domain, and

wherein

the reactivity of a PS-DH domain with 3-hydroxyhexanoyl ACP in the microorganism is higher than the reactivity of a FabA-DH domain with 3-hydroxyhexanoyl ACP in the microorganism

wherein the introduced genes are one of the following combinations:

(i) phoA, phoB, phoC, phoD, epaE, araB,

(ii) epaA, phoB, phoC, phoD, epaE, araB,

(iii) epaA, araB, araC, araD, epaE,

(iv) phoA, araB, araC, araD, epaE, and

(v) orfA, orfB, orfC, epaE, araB, and

wherein

the amino acid sequence of the protein encoded by the arab gene is SEQ ID NO: 58,

the amino acid sequence of the protein encoded by the araC gene is SEQ ID NO: 60,

the amino acid sequence of the protein encoded by the araD gene is SEQ ID NO: 62,

the amino acid sequence of the protein encoded by the phoA gene is SEQ ID NO: 66,

the amino acid sequence of the protein encoded by the phoB gene is SEQ ID NO: 68,

the amino acid sequence of the protein encoded by the phoC gene is SEQ ID NO: 70,

the amino acid sequence of the protein encoded by the phoD gene is SEQ ID NO: 72,

the amino acid sequence of the protein encoded by the epaA gene is SEQ ID NO: 74,

the amino acid sequence of the protein encoded by the epaE gene is SEQ ID NO: 82,

the amino acid sequence of the protein encoded by the orfA gene is SEQ ID NO: 84,

the amino acid sequence of the protein encoded by the orfB gene is SEQ ID NO: 86 or 90, and

the amino acid sequence of the protein encoded by the orfC gene is SEQ ID NO: 88 or 92.

6. The microorganism according to claim 5 , which possesses all PUFA synthetic pathway genes possessed by the microorganism capable of producing a ω-6 PUFA, wherein the reactivity of the PS-DH domain with 3-hydroxyhexanoyl ACP in the microorganism is higher than the reactivity of a FabA-DH domain with 3-hydroxyhexanoyl ACP in the microorganism.

7. The microorganism according to claim 5 , wherein the ω-6 PUFA produced is ARA or DPA.

8. The microorganism according to claim 5 , wherein the microorganism that does not have a PUFA 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.

9. A method for producing a ω-6 PUFA or a ω-6 PUFA-containing composition, comprising:

culturing the microorganism according to claim 1 in a culture medium so as to produce and accumulate a ω-6 PUFA or a ω-6 PUFA-containing composition in a culture, and

collecting the ω-6 PUFA or the ω-6 PUFA-containing composition from the culture.

10. A method for producing a ω-6 PUFA or a ω-6 PUFA-containing composition using the following microorganism (I) or (II) capable of producing a ω-6 PUFA:

(I) a microorganism capable of producing a ω-6 PUFA, in which the reactivity of a PS-DH domain with 3-hydroxyhexanoyl ACP in the microorganism is higher than the reactivity of a FabA-DH domain with 3-hydroxyhexanoyl ACP in the microorganism,

wherein the microorganism has an anaerobic pathway involving a protein complex having PUFA-PKS activity,

wherein proteins constituting the protein complex having PUFA-PKS activity are one of the following combinations (i) to (v):

(i) PhoA, PhoB, PhoC, PhoD, EpaE, AraB,

(ii) EpaA, PhoB, PhoC, PhoD, EpaE, AraB,

(iii) EpaA, AraB, AraC, AraD, EpaE,

(iv) PhoA, AraB, AraC, AraD, EpaE, and

(v) OrfA, OrfB, OrfC, EpaE, AraB, and

wherein

the amino acid sequence of AraB is SEQ ID NO: 58,

the amino acid sequence of AraC is SEQ ID NO: 60,

the amino acid sequence of AraD is SEQ ID NO: 62,

the amino acid sequence of PhoA is SEQ ID NO: 66,

the amino acid sequence of PhoB is SEQ ID NO: 68,

the amino acid sequence of PhoC is SEQ ID NO: 70,

the amino acid sequence of PhoD is SEQ ID NO: 72,

the amino acid sequence of EpaA is SEQ ID NO: 74,

the amino acid sequence of EpaE is SEQ ID NO: 82,

the amino acid sequence of OrfA is SEQ ID NO: 84,

the amino acid sequence of OrfB is SEQ ID NO: 86 or 90, and

the amino acid sequence of OrfC is SEQ ID NO: 88 or 92,

or

(II) a microorganism capable of producing a ω-6 PUFA, in which genes encoding respective domains described in the following (a) to (j) having a PUFA-PKS activity have been introduced into a microorganism that does not have a PUFA metabolic pathway:

(a) a KS domain;

(b) a MAT domain;

(c) an ACP domain;

(d) a KR domain;

(e) a PS-DH domain;

(f) a CLF domain;

(g) an AT domain;

(h) a FabA -DH domain;

(i) an ER domain; and

(j) a PPT domain,

wherein the reactivity of a PS-DH domain with 3-hydroxyhexanoyl ACP in the microorganism is higher than the reactivity of a FabA-DH domain with 3-hydroxyhexanoyl ACP in the microorganism

wherein the introduced genes are one of the following combinations:

(i) phoA, phoB, phoC, phoD, epaE, araB,

(ii) epaA, phoB, phoC, phoD, epaE, araB,

(iii) epaA, araB, araC, araD, epaE,

(iv) phoA, araB, araC, araD, epaE, and

(v) orfA, orfB, orfC, epaE, araB, and

wherein

the amino acid sequence of the protein encoded by the arab gene is SEQ ID NO: 58,

the amino acid sequence of the protein encoded by the araC gene is SEQ ID NO: 60,

the amino acid sequence of the protein encoded by the araD gene is SEQ ID NO: 62,

the amino acid sequence of the protein encoded by the phoA gene is SEQ ID NO: 66,

the amino acid sequence of the protein encoded by the phoB gene is SEQ ID NO: 68,

the amino acid sequence of the protein encoded by the phoC gene is SEQ ID NO: 70,

the amino acid sequence of the protein encoded by the phoD gene is SEQ ID NO: 72,

the amino acid sequence of the protein encoded by the epaA gene is SEQ ID NO: 74,

the amino acid sequence of the protein encoded by the epaE gene is SEQ ID NO: 82,

the amino acid sequence of the protein encoded by the orfA gene is SEQ ID NO: 84,

the amino acid sequence of the protein encoded by the orfB gene is SEQ ID NO: 86 or 90, and

the amino acid sequence of the protein encoded by the orfC gene is SEQ ID NO: 88 or 92.

11. A method for producing a ω-6 PUFA or a ω-6 PUFA-containing composition, comprising:

culturing the microorganism according to claim 5 in a culture medium so as to produce and accumulate a ω-6 PUFA or a ω-6 PUFA-containing composition in a culture, and

collecting the ω-6 PUFA or the ω-6 PUFA-containing composition from the culture.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 8, 2021
From: DAIRI, TOHRU; SATOH, YASUHARU; HAYASHI, SHOHEI; OGASAWARA, YASUSHI; UJIHARA, TETSURO
To: KYOWA HAKKO BIO CO., LTD.
Reel/Frame 055187/0699 →
Priority Claims (1)
JP 2018-151233 · Aug 10, 2018 · national
Continuity (1)
Related Publication 20210309987A1 · Oct 7, 2021
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