# US 2019/0169661 A1 · Genes and proteins for aromatic polyketide synthesis

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- Number: US 2019/0169661 A1
- Document ID: US20190169661A1
- Status: Published application
- Kind: A1
- Application number: 16/051,701
- Filed: 2018-08-01
- Published: 2019-06-06
- Earliest claimed: 2010-04-15
- Assignees: National Research Council of Canada; University of Saskatchewan
- Applicants: National Research Council of Canada; University of Saskatchewan
- Inventors: Jonathan E. Page; Steve Gagne
- Web page: https://patentgrep.com/patent/US20190169661A1

## CPC

- C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein
- C12N 9/1025: {Acyltransferases (2.3)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- C12P 7/22: aromatic
- C12P 7/42: Hydroxy-carboxylic acids
- C12Y 203/01206: 3,5,7-Trioxododecanoyl-CoA synthase (2.3.1.206)
- C12Y 404/01026: Olivetolic acid cyclase (4.4.1.26)
- 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

Nucleic acid molecules encoding polypeptides having polyketide synthase activity have been identified and characterized. Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds in organisms. The polypeptides may be used in vivo or in vitro to produce cannabinoid compounds.

## Claims

### Claim at position 1

1-27. (canceled)

### Claim 28

A process of synthesizing a naturally-occurring cannabinoid compound or a non-naturally occurring analog of a cannabinoid compound in a microorganism comprising expressing a nucleic molecule that encodes a polypeptide with at least 95% sequence identity to SEQ ID NO: 2 or that is a conservatively substituted amino acid sequence of the sequence set forth in SEQ ID NO: 2 and having polyketide cyclase activity, in the microorganism in the presence of a type III polyketide synthase enzyme, an alkanoyl CoA and malonyl CoA.

### Claim 29 (depends on 28)

The process of claim 28, wherein the microorganism is a yeast or a bacteria.

### Claim 30 (depends on 28)

The process of claim 28, wherein the microorganism is Saccharomyces cerevisiae yeast or E. coli.

### Claim 31 (depends on 28)

The process of claim 28, wherein the nucleic acid molecule is expressed or over-expressed in combination with expression or over-expression of one or more other nucleic acids that encode one or more enzymes in a cannabinoid biosynthetic pathway.

### Claim 32 (depends on 31)

The process of claim 31, wherein the one or more enzymes in a cannabinoid biosynthetic pathway is one or more of an acyl CoA synthetase, a polyketide cyclase, an aromatic prenyltransferase or a cannabinoid-forming oxidocylase.

### Claim 33 (depends on 32)

The process of claim 32, wherein the one or more enzymes in a cannabinoid biosynthetic pathway is one or more of a hexanoyl CoA synthetase, a geranylpyrophosphate:olivetolate geranyltransferase, a Δ9-tetrahydrocannabinolic acid synthase, a cannabidiolic acid synthase or a cannabichromenic acid synthase.

### Claim 34 (depends on 28)

The process of claim 28, wherein the cannabinoid compound is one or more of cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, Δ9-tetrahydrocannabinol, cannabidiol or cannabichromene or an analog thereof comprising a side-chain of 1 to 9 carbon atoms in length.

### Claim 35 (depends on 28)

The process of claim 28, wherein the alkanoyl CoA is CoA-S—C(O)—(CH2)n-CH3, where n is an integer from 0 to 4.

### Claim 36 (depends on 35)

The process of claim 35, wherein the alkanoyl CoA comprises hexanoyl CoA.

### Claim 37 (depends on 28)

The process of claim 28, wherein the type III polyketide synthase enzyme is polyketide synthase/olivetol synthase.

### Claim 38 (depends on 28)

The process of claim 28, wherein the nucleic acid molecule has a sequence of SEQ ID NO: 1 or 3.

### Claim 39

A process of synthesizing a polyketide in an in vitro cell-free reaction, said process comprising:
reacting an acyl carboxylic acid with coenzyme A through the action of an acyl CoA synthetase to form an alkanoyl CoA in presence of a type III polyketide synthase enzyme and a polypeptide with at least 95% sequence identity to SEQ ID NO: 2 or that is a conservatively substituted amino acid sequence of the sequence set forth in SEQ ID NO: 2 and having polyketide cyclase activity.

### Claim 40 (depends on 39)

The process of claim 39, further comprises reacting the alkanoyl CoA with a malonyl CoA.

### Claim 41 (depends on 40)

The process of claim 40, wherein the alkanoyl CoA is CoA-S—C(O)—(CH2)n—CH3, where n is an integer from 0 to 6.

### Claim 42 (depends on 41)

The process of claim 41, wherein the alkanoyl CoA comprises hexanoyl CoA, butyryl CoA, or octanoyl CoA.

### Claim 43 (depends on 40)

The process of claim 40, wherein the type III polyketide synthase enzyme is polyketide synthase/olivetol synthase.

### Claim 44 (depends on 42)

The process of claim 42, wherein the alkanoyl CoA is hexanoyl CoA.

### Claim 45 (depends on 44)

The process of claim 44, wherein the polyketide is olivetolic acid.

### Claim 46 (depends on 42)

The process of claim 42, wherein the alkanoyl CoA is butyryl CoA

### Claim 47 (depends on 42)

The process of claim 42, wherein the alkanoyl CoA is octanoyl CoA.