# US 11,898,190 B2 · Labyrinthulid microorganism capable of producing microbial oil, microbial oil, methods for producing said microorganism and for producing said microbial oil, and uses of said microorganism and said microbial oil

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- Number: US 11,898,190 B2
- Document ID: US11898190B2
- Status: Granted
- Kind: B2
- Application number: 17/401,894
- Filed: 2021-08-13
- Published: 2022-02-10
- Granted: 2024-02-13
- Earliest claimed: 2015-07-03
- Assignees: Kyushu University, National University Corporation; Konan Gakuen; Nippon Suisan Kaisha, Ltd.
- Applicants: Kyushu University, National University Corporation; Konan Gakuen; Nippon Suisan Kaisha, Ltd.
- Inventors: Yuji Okita; Makoto Ito; Rie Hamaguchi; Hatsumi Goda; Seiya Mochinaga; Daisuke Honda
- Web page: https://patentgrep.com/patent/US11898190B2

## CPC

- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12P 7/6432: Eicosapentaenoic acids [EPA]
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 15/09: Recombinant DNA-technology
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12P 1/00: Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P 7/64: 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
- C12P 7/6427: Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P 7/6472: containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Y 114/19: with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
- C12Y 602/01003: Long-chain-fatty-acid-CoA ligase (6.2.1.3)
- C10G 3/00: Production of liquid hydrocarbon mixtures from oxygen-containing organic materials, e.g. fatty oils, fatty acids (production from non-melting solid oxygen-containing carbonaceous materials C10G1/00)
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N 1/125: Unicellular algae isolates
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12R 2001/00: Microorganisms {; Processes using microorganisms}
- C12R 2001/89: Algae {; Processes using algae}
- Y02P 30/20: using bio-feedstock
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- C10G: CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION (cracking to hydrogen or synthesis gas C01B; cracking or pyrolysis of hydrocarbon gases to individual hydrocarbons or mixtures thereof of definite or specified constitution C07C; cracking to cokes C10B); RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

A method for producing a microbial oil includes steps of: genetically modifying a labyrinthulid by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene, and culturing the labyrinthulid, such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content; and collecting the microbial oil having the increased EPA content from the labyrinthulid. The labyrinthulid before the modification is selected from (A) a labyrinthulid belonging to the genus Parietichytrium or genus Schizochytrium and having very weak or no activity of producing PUFAs via a PUFA-PKS pathway; and (B) a labyrinthulid belonging to the genus Thraustochytrium in which a host PUFA-PKS gene is disrupted or silenced to a very weak level. The increased EPA content is preferably not less than 11.5% of a total fatty acid composition.

## Claims

### Claim 1

A method for producing a microbial oil, comprising:
genetically modifying a labyrinthulid by disrupting and/or silencing a fatty acid desaturase gene;
culturing the labyrinthulid; and
collecting the microbial oil from the labyrinthulid,
wherein the labyrinthulid before the modification belongs to the genus Parietichytrium or the genus Schizochytrium having an activity of synthesizing DHA via a PUFA-PKS pathway in an amount of not more than 1/100 of a total amount of DHA synthesized in the labyrinthulid or no activity of producing PUFAs via the PUFA-PKS pathway, and having an activity of producing PUFAs via an endogenous elongase-desaturase pathway,
wherein the fatty acid desaturase gene is a Δ4 desaturase gene,
wherein the microbial oil satisfies (a) and (b):
(a) DHA is not greater than 0.50% of the total fatty acid composition; and
(b) a total of DHA and n-6 DPA is not greater than 0.7% of the total fatty acid composition.

### Claim 2 (depends on 1)

The method for producing microbial oil according to claim 1,
wherein the step of disrupting the gene of the labyrinthulid utilizes electroporation, a gene gun method, or gene editing, and/or wherein the step of gene silencing of a labyrinthulid utilizes an antisense method or RNA interference.

### Claim 3 (depends on 1)

The method for producing microbial oil according to claim 1, wherein the labyrinthulid belonging to the genus Parietichytrium is Parietichytrium sarkarianum, and/or wherein the labyrinthulid belonging to the genus Schizochytrium is Schizochytrium aggregatum.

### Claim 4 (depends on 1)

The method for producing microbial oil according to claim 1,
wherein the labyrinthulid belonging to the genus Parietichytrium is Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sarkarianum SEK364 (FERM BP-11298), or Parietichytrium sp. SEK571 (FERM BP11406), and/or wherein the labyrinthulid belonging to the genus Schizochytrium is Schizochytrium aggregatum ATCC 28209.