# US 11,306,317 B2 · Method of producing isoprenoids and proteins, genes, and transformants for the same

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- Number: US 11,306,317 B2
- Document ID: US11306317B2
- Status: Granted
- Kind: B2
- Application number: 16/612,413
- Filed: 2018-05-11
- Published: 2020-02-27
- Granted: 2022-04-19
- Earliest claimed: 2017-05-11
- Assignees: Nagasaki University; Kikkoman Corporation
- Applicants: Kikkoman Corporation; Nagasaki University
- Inventors: Yasuko Araki; Yasutomo Shinohara; Kiyoshi Kita
- Web page: https://patentgrep.com/patent/US11306317B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C07K 14/37: from fungi
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 9/0004: {Oxidoreductases (1.)}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/0073: {with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13}
- C12N 9/1085: {transferring alkyl or aryl groups other than methyl groups (2.5)}
- C12N 9/14: Hydrolases (3)
- C12N 9/88: Lyases (4.)
- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12P 7/24: containing a carbonyl group
- C12P 7/26: Ketones
- C12P 17/04: containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C}
- C12Y 114/00: Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The problem to be solved by the present invention is to provide a method of producing isoprenoids including ascofuranone, ilicicolin A, and ascochlorin and derivatives thereof in a high yield as compared to the conventional art, which method enables industrial-scale production of isoprenoids. The problem can be solved by a method of producing isoprenoids such as ascofuranone, ilicicolin A, and ascochlorin, including using a transformant obtained by transformation with biosynthetic genes for ascofuranone, ilicicolin A, or ascochlorin or a knockout organism for these genes to obtain isoprenoids such as ascofuranone, ilicicolin A, and ascochlorin.

## Claims

### Claim 1

A transformant comprising an isolated gene inserted thereinto, and expressing the isolated gene, provided that the transformant is transformed isoprenoid-producing filamentous fungi, wherein the isolated gene comprises any one of nucleotide sequences of (1) to (4) below that encode an amino acid sequence of an enzyme having an activity of catalyzing monooxygenation of ilicicolin A epoxide:
(1) a nucleotide sequence set forth in SEQ ID NO: 8;
(2) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 8;
(3) a nucleotide sequence encoding an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 18;
and
(4) a nucleotide sequence encoding an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted, substituted, and/or added in the amino acid sequence set forth in SEQ ID NO: 18, provided that the amino acids are those per unit when 100 amino acids in the amino acid sequence are considered as one unit.

### Claim 2 (depends on 1)

The transformant of claim 1, wherein the isolated gene comprises a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 8.

### Claim 3 (depends on 1)

The transformant of claim 1, wherein the isolated gene comprises a nucleotide sequence encoding an amino acid sequence having 95% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

### Claim 4 (depends on 1)

The transformant of claim 1, wherein the isolated gene comprises a nucleotide sequence encoding an amino acid sequence having 1, 2, 3, 4, or 5 amino acids deleted, substituted, and/or added in the amino acid sequence set forth in SEQ ID NO: 18, provided that the amino acids are those per unit when 100 amino acids in the amino acid sequence are considered as one unit.

### Claim 5 (depends on 1)

The transformant of claim 1, wherein the isolated gene comprises a nucleotide sequence encoding an amino acid sequence having 1 or 2 amino acids deleted, substituted, and/or added in the amino acid sequence set forth in SEQ ID NO: 18, provided that the amino acids are those per unit when 100 amino acids in the amino acid sequence are considered as one unit.

### Claim 6 (depends on 1)

A method for producing ascofuranone, comprising:
providing the transformant according to claim 1,
growing the transformant, and
isolating ascofuranone from the transformant.