# US 10,767,187 B2 · Identification and characterization of UDP-glucose:phloretin 4′-O-glucosyl transferase from Malus x domestica Borkh

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- Number: US 10,767,187 B2
- Document ID: US10767187B2
- Status: Granted
- Kind: B2
- Application number: 16/082,623
- Filed: 2017-03-09
- Published: 2019-02-28
- Granted: 2020-09-08
- Earliest claimed: 2016-03-09
- Assignees: The State of Israel, Ministry of Agriculture & Rural Development, Agricultural Research Organization (ARO) (Volcani Center)
- Applicants: The State of Israel, Ministry of Agriculture & Rural Development Agricultural Research Organization (ARO) (Volcani Center)
- Inventors: Mwafaq Ibdah
- Web page: https://patentgrep.com/patent/US10767187B2

## CPC

- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- A61K 31/7034: attached to a carbocyclic compound, e.g. phloridzin
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 35/00: Antineoplastic agents
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)}
- C12Y 204/01: Hexosyltransferases (2.4.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

## Abstract

A method of producing trilobatin is disclosed. The method comprising contacting a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8 and having a 4′-O-glycosyltransferase activity with phloretin and UDP-glucose under conditions which allow the formation of trilobatin, thereby producing trilobatin. A method of producing a plant which produces trilobatin, transgenic plant or plant cell and methods of producing transgenic plants are disclosed. Composition comprising trilobatin are also disclosed.

## Claims

### Claim 1

A method of producing trilobatin, the method comprising contacting a polypeptide comprising an amino acid sequence at least 98% identical to SEQ ID NO: 8 and having a 4′-0-glycosyltransferase activity with phloretin and UDP-glucose under conditions which allow the formation of trilobatin, thereby producing trilobatin, and purifying said trilobatin, wherein said polypeptide is exogenously expressed in a plant cell, a bacterial or a yeast cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the formation of trilobatin is effected in vivo.

### Claim 3 (depends on 2)

The method of claim 2, wherein said in vivo is in a plant cell.

### Claim 4 (depends on 1)

The method of claim 1, wherein said amino acid sequence is at least 99% identical to SEQ ID NO: 8.

### Claim 5 (depends on 1)

The method of claim 1, wherein said amino acid sequence is SEQ ID NO: 8.

### Claim 6 (depends on 2)

The method of claim 2, wherein said in vivo is in a bacterial or yeast cell.

### Claim 7

A method of producing trilobatin, the method comprising transforming a plant cell, a bacterial cell or a yeast cell, with a polynucleotide encoding a polypeptide comprising an amino acid sequence at least 98% identical to SEQ ID NO: 8 and having a 4′-0-glycosyltransferase activity, thereby exogenously expressing said polypeptide in a plant cell, contacting said polypeptide with phloretin and UDP-glucose under conditions which allow the formation of trilobatin, producing trilobatin in said plant cell, bacterial cell or yeast cell, and purifying said trilobatin.