Metabolically engineered cells for the production of polyunsaturated fatty acids

• US 20060051847A1
• Filed: 06/06/2005
• Published: 03/09/2006
• Est. Priority Date: 06/04/2004
• Status: Active Grant

• Current Assignee: Fluxome Sciences A/S
• Original Assignee: Fluxome Sciences A/S
• Inventors: Forster, Jochen, Nielsen, Jens Bredal, Gunnarsson, Nina Katarina
• Granted Patent: US 7,736,884 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A61K 8/361 Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
• CPC Class Codes: A61K 8/67 Vitamins
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6431 Linoleic acids [18:2[n-6]]
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6458 by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6481 Phosphoglycerides phosphoglycerides having carboxylic acids with less than seven carbon atoms C12P7/62
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

The present invention relates to the construction and engineering of cells, more particularly microorganisms for producing PUFAs with four or more double bonds from non-fatty acid substrates through heterologous expression of an oxygen requiring pathway. The invention especially involves improvement of the PUFA content in the host organism through fermentation optimization, e.g. decreasing the temperature and/or designing an optimal medium, or through improving the flux towards fatty acids by metabolic engineering, e.g. through over-expression of fatty acid synthases, over-expression of other enzymes involved in biosynthesis of the precursors for PUFAs, or codon optimization of the heterologous genes, or expression of heterologous enzymes involved in the biosynthesis of the precursor for PUFAs.

Claims

1. A method for producing polyunsaturated fatty acids with four or more double bonds comprising heterologous expression of an oxygen requiring pathway in a Saccharomyces cerevisiae grown on a non-fatty acid substrate.

2. A method according to claim 1, wherein said non-fatty acid substrate is the exclusive carbon source.

3. A method according to claim 1, wherein the heterologous expression comprises combining heterologous expression of nucleotide sequences encoding delta-12 desaturase, delta-6 desaturase, delta-9 elongase, delta-8 desaturase, delta-6 elongase, and delta-5 desaturase, delta-5 elongase, omega-3 desaturase, delta-9 desaturase, and/or delta-4 desaturase.

4. A method according to claim 3, wherein said combined heterologous expression further comprises an over-expression of at least one of the genes selected from the group consisting of ACC1, YBR159W, ELO1, ELO2, ELO3, FAS1, FAS2, DGA1, LRO1, ARE1, ARE2, TSC13, GAT1, SLC1, YDR531W, and GPD1;

or

wherein said combined heterologous expression further comprises a deletion of at least one of the genes selected from the group consisting of GPP1, GPP2 GDH1 and POX1, and optionally an over-expression of at least one of the genes selected from the group consisting of GDH2, GLN1, and GLT1;

or

wherein said combined heterologous expression further comprises a heterologous expression of the nucleotide sequences encoding ATP;

citrate lyase, a heterologous expression of a nucleotide sequence encoding a non-phosphorylating NADP-dependent D-glyceraldehyde-3-phosphate dehydrogenase, and/or an isocitrate dehydrogenase which is stimulated by AMP

5. A method according to claim 1, wherein said heterologous nucleotide sequences are codon optimized for expression in Saccharomyces cerevisiae;

or

wherein said Saccharomyces cerevisiae is cultivated in a myo-inositol deficient medium.

6. A method according to claim 1, wherein the polyunsaturated fatty acid is selected from the group consisting of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.

7. A method according to claim 1, wherein said heterologous expression increases the content of arachidonic acid, eicosapentaenoic acid and/or docosahexaenoic acid to more than 2% of the total fatty acid content in said Saccharomyces cerevisiae

8. A method according to claim 3, wherein the nucleotide sequence encoding delta-12 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NOs 5-10, 93, 95, and 113;

and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NOs 5-10, 93, 95, and 113.

9. A method according to claim 3, wherein the nucleotide sequence encoding delta-9 elongase is a nucleotide sequence comprising or having at least 75% identity to the nucleotide sequence of SEQ ID NO:

37;

or

wherein the nucleotide sequence encoding delta-9 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

1-4; and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

1-4;

or

wherein the nucleotide sequence encoding delta-8 desaturase is a nucleotide sequence comprising or having at least 75% identity to the nucleotide sequence of SEQ ID NO;

38;

or

wherein the nucleotide sequence encoding delta-6 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

11-15, 97, and 99; and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

11-15, 97, and 99;

or

wherein the nucleotide sequence encoding delta-6 elongase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

16-21, 101 and 103; and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

16-21, 101 and 103.

10. A method according to claim 3, wherein the nucleotide sequence encoding delta-5 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO:

22-27, 99, 105 and 107; and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

22-27, 99, 105 and 107;

or

wherein the nucleotide sequence encoding delta-5 elongase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

19, 28, 29 and 101;

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

19, 28, 29 and 101; and

c) nucleotide sequences encoding amino acid sequences that have at least 75% identity to SEQ ID NO 68;

or

wherein the nucleotide sequence encoding delta-4 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

35-36 and 109;

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

35-36 and 109; and

c) nucleotide sequences encoding amino acid sequences that have at least 75% identity to SEQ ID NOs 76-77;

or

wherein the nucleotide sequence encoding omega-3 desaturase is selected from the group consisting of

a) the nucleotide sequences set forth in SEQ ID NO;

30-34, 87, 89 and 111; and

b) nucleotide sequences having at least 75% identity to the nucleotide sequences set forth in SEQ ID NO;

30-34, 87, 89 and 111.

11. A method for producing a polyunsaturated fatty acid comprising the steps of

(a) isolating at least 4 nucleotide sequences encoding delta-12 desaturase, delta-6 desaturase, delta-6 elongase, and delta-5 desaturase

(b) constructing one or more vectors comprising said isolated nucleotide sequences of step (a) and/or integrating said isolated nucleotide sequences into the genome of Saccharomyces cerevisiae;

(c) optionally, transforming said vector(s) of step (b) into a Saccharomyces cerevisiae for a time and under conditions sufficient for expression of proteins encoded by said isolated nucleotide sequences of step (a);

(d) growing said Saccharomyces cerevisiae on a non-fatty acid substrate, whereby said non-fatty acid substrate is converted by said host into a desired polyunsaturated fatty acid product

and obtaining said polyunsaturated fatty acid.

12. A method for producing a polyunsaturated fatty acid comprising the steps of

(a) isolating at least 4 nucleotide sequences encoding delta-12 desaturase, delta-9 elongase, delta-8 desaturase, and delta-5 desaturase

(b) constructing one or more vectors comprising said isolated nucleotide sequences of step (a) and/or integrating said isolated nucleotide sequences into the genome of Saccharomyces cerevisiae;

(c) optionally, transforming said vector(s) of step (b) into a Saccharomyces cerevisiae for a time and under conditions sufficient for expression of proteins encoded by said isolated nucleotide sequences of step (a);

(d) growing said Saccharomyces cerevisiae on a non-fatty acid substrate, whereby said non-fatty acid substrate is converted by said host into a desired polyunsaturated fatty acid product

and obtaining said polyunsaturated fatty acid.

14. A genetically modified Saccharomyces cerevisiae capable of producing polyunsaturated fatty acids with four or more double bonds when grown on a non-fatty acid substrate;

or wherein said Saccharomyces cerevisiae is capable of producing polyunsaturated fatty acids with four or more double bonds rown on a non-fatty acid substrate as the exclusive carbon source.

13. A method according to claim 18 or 19, wherein the heterologous expression further comprises heterologous expression of a nucleotide sequence encoding delta-9 desaturase, delta-5 elongase, omega-3 desaturase, and/or delta-4 desaturase.

15. A composition comprising a polyunsaturated fatty acid produced by a genetically modified Saccharomyces cerevisiae according to claim 14.

16. A composition comprising at least 25% polyunsaturated fatty acid in total fatty acid composition produced by a genetically modified cell according to claim 14.

17. A composition according to claim 15, wherein said composition is an oil;

or wherein said polyunsaturated fat is incorporated in triacylglycerides;

or wherein said polyunsaturated fatty acids is incorporated in phospholipids;

or wherein said polyunsaturated fatty acids is in a form of free fatty acids.

18. Use of a composition according to claim 15 as an ingredient in a food product;

or as an ingredient in a cosmetic product;

or as an ingredient in feed.

19. Use of a genetically modified Saccharomyces cerevisiae according to claim 14 as an ingredient in feed.