# US 2013/0160169 A1 · Fungal desaturase and elongase genes

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- Number: US 2013/0160169 A1
- Document ID: US20130160169A1
- Status: Published application
- Kind: A1
- Application number: 13/330,158
- Filed: 2011-12-19
- Published: 2013-06-20
- Earliest claimed: 2011-12-19
- Assignees: University of Saskatchewan; The Governors of the University of Alberta
- Inventors: Xiao QIU
- Web page: https://patentgrep.com/patent/US20130160169A1

## CPC

- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/001: {acting on the CH-CH group of donors (1.3)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12P 7/6427: Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P 7/6431: Linoleic acids [18:2[n-6]]
- C12P 7/6432: Eicosapentaenoic acids [EPA]
- C12Y 114/19003: Linoleoyl-CoA desaturase (1.14.19.3)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The invention is directed to isolated polynucleotide and polypeptides of the CoD5, CoD6 and CoE6 genes from Conidiobolus obscurus, the CtE6 gene from Conidiobolus thromboids, and the PgDesX gene from Puccinia graminis; nucleic acid constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.

## Claims

### Claim 1

An isolated polynucleotide encoding a polypeptide having Δ5, Δ6, or ω-3 desaturase activity or Δ6 elongase activity and comprising:
(a) an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8 or 10; or
(b) an amino acid sequence having at least 85% sequence identity with one of SEQ ID NO: 2, 4, 6, 8 or 10.

### Claim 2 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having Δ5 desaturase activity and comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having Δ5 desaturase activity and having at least 85% sequence identity therewith.

### Claim 3 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having Δ6 desaturase activity and comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having Δ6 desaturase activity and having at least 85% sequence identity therewith.

### Claim 4 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having ω-3 desaturase activity and comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having ω-3 desaturase activity and having at least 85% sequence identity therewith.

### Claim 5 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a polypeptide having Δ6 elongase activity and comprising the amino acid sequence of SEQ ID NO: 6 or 8, or an amino acid sequence having Δ6 elongase activity and having at least 85% sequence identity with one of SEQ ID NO: 6 or 8.

### Claim 6 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7 or 9.

### Claim 7 (depends on 1)

The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one of SEQ ID NO: 2, 4, 6, 8 or 10.

### Claim 8 (depends on 1)

The isolated polynucleotide of claim 1, wherein the polynucleotide is derived from Conidiobolus obscurus, Conidiobolus thromboids, or Puccinia graminis.

### Claim 9 (depends on 1)

A polynucleotide construct or vector comprising a polynucleotide of claim 1 operably linked to a promoter expressible in bacterial, yeast, fungal, mammalian or plant cells.

### Claim 10 (depends on 9)

A microbial cell comprising a polynucleotide construct or vector of claim 9.

### Claim 11 (depends on 10)

The microbial cell of claim 10, wherein the cell is Saccharomyces cerevisiae, Aspergillus, Pichia pastoris, Escherichia coli or Bacillus subtilis.

### Claim 12 (depends on 11)

The microbial cell of claim 11 which comprises a heterologous eicosatetraenoic acid biosynthetic pathway comprising a Δ6 desaturase, a Δ6 elongase, a Δ12 desaturase and an ω3 desaturase.

### Claim 13 (depends on 12)

The microbial cell of claim 12 which is S. cerevisiae and which comprises CoD6 and CoE6 from C. obscurus, CpDes12 and CpDesX from Claviceps purpurea.

### Claim 14 (depends on 9)

A transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising a polynucleotide construct or vector of claim 9.

### Claim 15 (depends on 14)

The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 14, which is selected from a linseed, canola, peanut, safflower, flax, oats, wheat, triticale, barley, corn, and legume plant, plant cell, plant seed, callus, plant embryo, or microspore-derived embryo or microspore.

### Claim 16 (depends on 1)

A method for producing a polyunsaturated fatty acid comprising the steps of:
a) constructing a vector comprising one or more polynucleotides of claim 1;
b) transforming the vector into a host cell under conditions sufficient for expression of a Δ5, Δ6 or ω-3 desaturase or a Δ6 elongase encoded by the polynucleotides; and
c) exposing the Δ5, Δ6, or ω-3 desaturase or A6 elongase to a fatty acid substrate, wherein the substrate is converted by the Δ5, Δ6, or ω-3 desaturase or A6 elongase into a desired polyunsaturated fatty acid product.

### Claim 17 (depends on 16)

The method of claim 16, wherein the fatty acid substrate comprises one or more of linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid.

### Claim 18 (depends on 17)

The method of claim 17, wherein the polypeptide comprises a Δ6 desaturase, and the fatty acid substrate comprises linoleic acid or α-linolenic acid.

### Claim 19 (depends on 17)

The method of claim 17, wherein the polypeptide comprises a Δ6 elongase, and the fatty acid substrate comprises γ-linolenic acid, stearidonic acid, arachidonic acid or eicosapentaenoic acid.

### Claim 20 (depends on 17)

The method of claim 17, wherein the polypeptide comprises an ω-3 desaturase, and the fatty acid substrate comprises linoleic acid, γ-linoleic acid, dihomo gamma-linoleic acid and arachidonic acid.