# US 2017/0253901 A1 · Method for producing a compound of the sterol biosynthesis pathway in a eukaryotic organism

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- Number: US 2017/0253901 A1
- Document ID: US20170253901A1
- Status: Published application
- Kind: A1
- Application number: 15/512,534
- Filed: 2015-09-16
- Published: 2017-09-07
- Earliest claimed: 2014-09-17
- Assignees: Institut National de la Recherche Agronomique; Centre International D'Etudes Superieures En Sciences Agronomiques; Universite De Montpellier; Centre National De La Recherche Scientifique
- Applicants: Institut National De La Recherche Agronomique; Centre International D'Etudes Superieures En Sciences Agronomiques; Universite De Montpellier; Centre National De La Recherche Scientifique
- Inventors: PIERRE BERTHOMIEU; ORIANE MITH; LAURENCE MARQUES-BASTIDE; VÉRONIQUE PERRIER; ERIC DUBREUCQ
- Web page: https://patentgrep.com/patent/US20170253901A1

## CPC

- C12P 33/00: Preparation of steroids
- C07K 14/415: from plants
- C12N 1/18: Baker's yeast; Brewer's yeast
- C12N 1/38: Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence)
- 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)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention concerns a method for producing a compound of interest of the sterol biosynthesis pathway in a eukaryotic organism or a derivative of a compound of the sterol biosynthesis pathway in said eukaryotic organism, in particular a sterol. This method comprises a step for the in vitro culturing of cells of said eukaryotic organism modified to express or overexpress a defensin, in a culture medium suitable for the development of said cells and containing at least one element chosen from the transition metals, lead and selenium, at a concentration greater than or equal to the concentration of said element that is necessary for inducing the overproduction of said compound of interest by said cells.

## Claims

### Claim 1

A method for producing a compound of interest of a sterol biosynthesis pathway in a eukaryotic organism or derived from a compound of the sterol biosynthesis pathway in said eukaryotic organism, comprising a step of in vitro culture of cells of said eukaryotic organism, modified to express or overexpress a defensin, in an enriched culture medium configured to grow said cells and containing at least one element chosen from a group consisting of transition metals, lead and selenium, at a concentration greater than or equal to a concentration of said at least one element for inducing production by said cells of an amount of said compound of interest at least 2 times greater than an amount produced by said cells under identical culture conditions in a culture medium containing said at least one element at a minimum concentration for an optimal growth of said cells.

### Claim 2 (depends on 1)

The method as claimed in claim 1, wherein the defensin is a defensing of a plant origin encoded by a gene of a Plant Defensin type 1 family having at least one of a three-dimensional structure comprising a CSαβ type motif, a gamma-core loop, and at least 30% amino acid identity with a defensin AhPDF1.1b of Arabidopsis halleri of sequence SEQ ID NO:2.

### Claim 3 (depends on 1)

The method as claimed in claim 1, wherein said at least one element is zinc.

### Claim 4 (depends on 1)

The method as claimed in claim 1, wherein said at least one element is present in said enriched culture medium at a concentration greater than or equal to the concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least five times greater than the amount produced by said cells under identical culture conditions in said culture medium containing said at least one element at the minimum concentration for the optimal growth of said cells.

### Claim 5 (depends on 1)

The method as claimed in claim 1, wherein said eukaryotic organism is chosen from a group consisting of yeasts, fungi, algae, microalgae, plants and animals.

### Claim 6 (depends on 1)

The method as claimed in claim 1, wherein the cells of said eukaryotic organism are recombinant cells into which a nucleotide sequence coding for said defensin has been introduced.

### Claim 7 (depends on 1)

The method as claimed in claim 1, further comprising a subsequent step of collecting the cells of said eukaryotic organism having accumulated said compound of interest.

### Claim 8 (depends on 1)

The method as claimed in claim 1, wherein the compound of interest is a final compound in the sterol biosynthesis pathway.

### Claim 9 (depends on 1)

The method as claimed in claim 1, wherein the compound of interest is an intermediate compound in the sterol biosynthesis pathway and said cells of said eukaryotic organism are recombinant cells modified to inhibit an enzyme activity involved in a biotransformation of said intermediate compound in the sterol biosynthesis pathway.

### Claim 10 (depends on 1)

The method as claimed in claim 1, wherein the compound of interest is a compound derived from a compound of the sterol biosynthesis pathway, and said cells of said eukaryotic organism are recombinant cells modified to activate an enzyme activity involved in a biotransformation of said compound of the sterol biosynthesis pathway into said compound of interest.

### Claim 11

A recombinant eukaryotic cell modified to express or overexpress a defensin and modified to inhibit an enzyme activity involved in a biotransformation of an intermediate compound in a sterol biosynthesis pathway in said recombinant eukaryotic cell.

### Claim 12 (depends on 11)

The recombinant eukaryotic cell as claimed in claim 11, modified to activate an enzyme activity involved in the biotransformation of said intermediate compound into a compound of interest.

### Claim 13

A method of using a recombinant yeast strain modified to express or overexpress a defensing for production of a compound of interest of a sterol biosynthesis pathway or derived from a compound of the sterol biosynthesis pathway in said recombinant yeast strain.

### Claim 14 (depends on 13)

The method as claimed in claim 13, wherein cells of said recombinant yeast strain are subjected to a step of in vitro culture in an enriched culture medium configured to grow said cells and containing at least one element chosen from a group consisting of transition metals, lead and selenium, at a concentration greater than or equal to a concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least 2 times greater than an amount produced by said cells under identical culture conditions in a culture medium containing said at least one element at a minimum concentration for an optimal growth of said cells.

### Claim 15 (depends on 13)

The method as claimed in claim 13, wherein the defensin is a defensing of a plant origin encoded by a gene of a Plant Defensin type 1 family having at least one of a three-dimensional structure comprising a CSαβ type motif, a gamma-core loop, and at least 30% amino acid identity with a defensin AhPDF1.1b of Arabidopsis halleri of sequence SEQ ID NO:2.

### Claim 16 (depends on 1)

The method as claimed in claim 1, comprising subsequent steps of collecting the cells of said eukaryotic organism having accumulated said compound of interest; and extracting said compound of interest.

### Claim 17 (depends on 10)

The method as claimed in claim 10, wherein said cells of said eukaryotic organism are said recombinant cells modified to inhibit the enzyme activity involved in the biotransformation of an intermediate compound in the sterol biosynthesis pathway.

### Claim 18 (depends on 4)

The method as claimed in claim 4, wherein said at least one element is present in said enriched culture medium at a concentration greater than or equal to the concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least 10 times greater than the amount produced by said cells under identical culture conditions in said culture medium containing said at least one element at the minimum concentration for the optimal growth of said cells.