# US 10,370,687 B2 · Genetically modified strain of eukaryotic microalga having improved triglyceride productivity, and use thereof

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- Number: US 10,370,687 B2
- Document ID: US10370687B2
- Status: Granted
- Kind: B2
- Application number: 15/756,233
- Filed: 2016-09-02
- Published: 2018-08-30
- Granted: 2019-08-06
- Earliest claimed: 2015-09-02
- Assignees: Chuo University; Denso Corporation
- Applicants: Chuo University; Denso Corporation
- Inventors: Shigeaki Harayama; Yoko Ide; Jun Abe; Yuki Kasai; Norihide Kurano
- Web page: https://patentgrep.com/patent/US10370687B2

## CPC

- C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/6445: Glycerides
- C12Y 203/0102: Diacylglycerol O-acyltransferase (2.3.1.20)
- C12Y 301/02014: Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
- C12Y 302/0102: Alpha-glucosidase (3.2.1.20)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

It is an object of the present invention to provide the eukaryotic microalgae, which have been genetically modified such that larger amounts of assimilation products produced by photosynthesis are directed to the synthesis of triglyceride (=triacylglycerol; TAG), and specifically, the present invention relates to a genetically modified strain of eukaryotic microalgae, in which a gene encoding an AGL1 protein is highly expressed, or a gene encoding an FAT1 protein and/or a gene encoding a DGAT2 protein are further highly expressed, as well as the gene encoding an AGL1 protein, wherein TAG productivity is improved in comparison to the parent strain thereof.

## Claims

### Claim 1

A genetically modified strain of eukaryotic microalgae, in which a gene encoding an alpha-glucosidase (AGL1) protein is highly expressed, wherein
triacylglycerol (TAG) productivity is improved in comparison to a parent strain thereof, and
the AGL1 protein has an amino acid sequence having sequence identity of at least 90% with the amino acid sequence shown in SEQ ID NO: 4, and has alpha-glucosidase activity.

### Claim 2 (depends on 1)

The genetically modified strain of eukaryotic microalgae according to claim 1, in which a gene encoding an acyl-ACP thioesterase (FAT1) protein and/or a gene encoding a diacylglycerol acyltransferase 2 (DGAT2) protein are also highly expressed, wherein
the FAT1 protein has an amino acid sequence having sequence identity of at least 90% with the amino acid sequence shown in SEQ ID NO: 8, and has acyl-ACP thioesterase activity, and the DGAT2 protein has an amino acid sequence having sequence identity of at least 90% with the amino acid sequence shown in SEQ ID NO: 12, and has diacylglycerol acyltransferase activity.

### Claim 3 (depends on 1)

The genetically modified strain of eukaryotic microalgae according to claim 1, wherein the genes are operably linked to a promoter ensuring the high expression of the genes.

### Claim 4 (depends on 1)

The genetically modified strain of eukaryotic microalgae according claim 1, which belongs to the class Trebouxiophyceae.

### Claim 5 (depends on 4)

The genetically modified strain of eukaryotic microalgae according to claim 4, which belongs to genus Coccomyxa or genus Pseudococcomyxa.

### Claim 6 (depends on 1)

The genetically modified strain of eukaryotic microalgae according to claim 1, wherein the genes are derived from a strain belonging to green algae.

### Claim 7 (depends on 6)

The genetically modified strain of eukaryotic microalgae according to claim 6, wherein the genes are derived from a strain belonging to the class Trebouxiophyceae.

### Claim 8 (depends on 7)

The genetically modified strain of eukaryotic microalgae according to claim 7, wherein the genes are derived from a strain belonging to genus Coccomyxa or genus Pseudococcomyxa.

### Claim 9 (depends on 1)

A method for producing TAG, comprising a step of culturing the genetically modified strain of eukaryotic microalgae according to claim 1.