ALGAL ELONGASE 6

Abstract

Provided herein are exemplary isolated nucleotide sequences encoding polypeptides having elongase activity, which utilize fatty acids as substrates.

Description

REFERENCE TO SEQUENCE LISTINGS

The present application is filed with sequence listing(s) attached hereto and incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to molecular biology, and more specifically, to algal elongases.

SUMMARY OF THE INVENTION

Isolated nucleotide sequences encoding polypeptides having elongase activity, which utilize fatty acids as substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence encoding elongase 1 (SEQ ID NO:1).

FIG. 2 illustrates the nucleotide sequence encoding elongase 2 (SEQ ID NO:2).

FIG. 3 illustrates the nucleotide sequence encoding elongase 3 (SEQ ID NO:3).

FIG. 4 illustrates the nucleotide sequence encoding elongase 4 (SEQ ID NO:4).

FIG. 5 illustrates the nucleotide sequence encoding elongase 5 (SEQ ID NO:5).

FIG. 6 illustrates the nucleotide sequence encoding elongase 6 (SEQ ID NO:6).

FIG. 7 illustrates the nucleotide sequence encoding elongase 7 (SEQ ID NO:7).

FIG. 8 illustrates the nucleotide sequence encoding elongase 8 (SEQ ID NO:8).

FIG. 9 illustrates the amino acid sequence encoding elongase 1 (SEQ ID NO:9).

FIG. 10 illustrates the amino acid sequence encoding elongase 2 (SEQ ID NO:10).

FIG. 11 illustrates the amino acid sequence encoding elongase 3 (SEQ ID NO:11).

FIG. 12 illustrates the amino acid sequence encoded by elongase 4 (SEQ ID NO:12).

FIG. 13 illustrates the amino acid sequence encoded by elongase 5 (SEQ ID NO:13).

FIG. 14 illustrates the amino acid sequence encoded by elongase 6 (SEQ ID NO:14).

FIG. 15 illustrates the amino acid sequence encoded by elongase 7 (SEQ ID NO:15).

FIG. 16 illustrates the amino acid sequence encoded by elongase 8 (SEQ ID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

A fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Saturated fatty acids are long-chain carboxylic acids that usually have between 12 and 24 carbon atoms and have no double bonds. Unsaturated fatty acids have one or more double bonds between carbon atoms. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Elongases are enzymes which lengthen fatty acids by adding two carbon atoms to a fatty acid's carboxylic acid end.

Provided herein are isolated nucleotide sequences encoding polypeptides having elongase activity, which utilize fatty acids as substrates.

The inventors sequenced the entire genome of algal genus Nannochloropsis and identified genes involved in fatty acid metabolism. They identified various elongases, including exemplary elongases which they designated as elongases 1-9.

The inventors manipulated the activities of the above-specified exemplary elongase genes by:

1. Overexpression of the subject elongase gene with a strong promoter.

2. Promoter replacement or promoter insertion in front of the subject elongase gene within the genome via homologous recombination.

3. Knock out of the subject elongase gene via insertion of a transformation construct into the gene or replacement of a part of or the entire subject elongase gene via homologous recombination.

Exemplary support for the above-mentioned methods may be found in U.S. Non-Provisional Patent Application Ser. No. 12/581,812 filed on Oct. 19, 2009, titled “Homologous Recombination in an Algal Nuclear Genome,” U.S. Non-Provisional Patent Application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” and U.S. Non-Provisional Patent Application Ser. No. 12/480,611 filed on Jun. 8, 2009, titled “Transformation of Algal Cells,” all of which are hereby incorporated by reference.

Accordingly, the inventors were able to manipulate the activities of the various exemplary elongases for the purpose of modifying the contents of certain fatty acids within algal genus Nannochloropsis.

Some of these elongases, i.e. Elongases 6-8, are down-regulated under conditions when poly unsaturated fatty acid (“PUFA”) biosynthesis is down-regulated as well (i.e. during Nitrogen starvation). These genes are excellent targets for over-expression, in order to achieve elevated PUFA biosynthesis. Down-regulation of these (or other) genes, as an example, by replacement of the endogenous promoter or insertion of a weaker promoter in front of the respective elongase gene could lead to a higher content of short chain fatty acids. Down-regulation of transcription could also be achieved, in some cases, by insertion of a commonly strong promoter in front of the respective elongase gene, presumably by modifying the respective chromatin arrangement around the said elongase gene, thus leading to a lower transcription level. Also, the introduction of point mutations into the gene when inserting another promoter in front of such a gene via the homologous recombination flanks utilized, could lead to an altered activity of the respective gene products.

Over expression and knock out mutants of said elongase genes suggest that at least 4 elongases with overlapping functions are operating in the biosynthesis pathway leading to Eicosapentaenoic acid (“EPA”): these are, but not limited to: Elongases 5, 6, 7, and 9. Transcriptome analysis also suggests that Elongase 8 is operating as well in the fatty acid biosynthesis pathway to EPA.

FIG. 1 illustrates the nucleotide sequence encoding elongase 1 (SEQ ID NO:1).

FIG. 2 illustrates the nucleotide sequence encoding elongase 2 (SEQ ID NO:2).

FIG. 3 illustrates the nucleotide sequence encoding elongase 3 (SEQ ID NO:3).

FIG. 4 illustrates the nucleotide sequence encoding elongase 4 (SEQ ID NO:4).

FIG. 5 illustrates the nucleotide sequence encoding elongase 5 (SEQ ID NO:5).

FIG. 6 illustrates the nucleotide sequence encoding elongase 6 (SEQ ID NO:6).

FIG. 7 illustrates the nucleotide sequence encoding elongase 7 (SEQ ID NO:7).

FIG. 8 illustrates the nucleotide sequence encoding elongase 8 (SEQ ID NO:8).

FIG. 9 illustrates the amino acid sequence encoding elongase 1 (SEQ ID NO:9).

FIG. 10 illustrates the amino acid sequence encoding elongase 2 (SEQ ID NO:10).

FIG. 11 illustrates the amino acid sequence encoding elongase 3 (SEQ ID NO:11).

FIG. 12 illustrates the amino acid sequence encoded by elongase 4 (SEQ ID NO:12).

FIG. 13 illustrates the amino acid sequence encoded by elongase 5 (SEQ ID NO:13).

FIG. 14 illustrates the amino acid sequence encoded by elongase 6 (SEQ ID NO:14).

FIG. 15 illustrates the amino acid sequence encoded by elongase 7 (SEQ ID NO:15).

FIG. 16 illustrates the amino acid sequence encoded by elongase 8 (SEQ ID NO:16).

While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of a preferred embodiment should not be limited by any of the above-described exemplary embodiments.

Claims

1-16. (canceled)

17. A vector comprising a polynucleotide sequence encoding elongase 6 having the amino acid sequence of SEQ ID NO:14.

18. The vector of claim 17, wherein the polynucleotide sequence is SEQ ID NO:6.

19. The vector of claim 17, further comprising a Violaxanthin-chlorophyll a binding protein (VCP) promoter.

20. The vector of claim 17, which is a transformation vector.

21. The vector of claim 17, which is a homologous recombination vector.

22. The vector of claim 17, which has been incorporated into the genome of an algal cell.

23. A method of modulating poly unsaturated fatty acid biosynthesis in an algal cell, the method comprising enhancing or suppressing the expression of elongase 6 having the amino acid sequence of SEQ ID NO:14.

24. The method of claim 23, wherein the expression of elongase 6 is enhanced and EPA production is increased in the algal cell.

25. The method of claim 23, wherein the expression of elongase 6 is suppressed and EPA production is decreased in the algal cell.

26. The method of claim 23, wherein the algal cell is a Nannochloropsis cell.

27. The method of claim 24, wherein the algal cell is present in a Nannochloropsis alga.

28. The method of claim 23, wherein the algal cell has been transformed with a stronger or weaker promoter in front of a genomic sequence encoding elongase 6 having the amino acid sequence of SEQ ID NO:14.

29. The method of claim 25, wherein the algal cell has been transformed to result in (1) an insertion in the genomic sequence encoding elongase 6 having the amino acid sequence of SEQ ID NO:14; or (2) a deletion or substitution of a portion or the full length of the genomic sequence encoding elongase 6.

30. A transformed algal cell with altered poly unsaturated fatty acid biosynthesis, wherein the algal cell has altered expression level of elongase 6 having the amino acid sequence of SEQ ID NO:14.

31. The algal cell of claim 30, wherein a stronger or weaker promoter is placed in front of a genomic sequence encoding elongase 6 having the amino acid sequence of SEQ ID NO:14.

32. The algal cell of claim 30, which has (1) an insertion in the genomic sequence encoding elongase 6 having the amino acid sequence of SEQ ID NO:14; or (2) a deletion or substitution of a portion or the full length of the genomic sequence encoding elongase 6.

33. The algal cell of claim 30, which is a Nannochloropsis cell.

34. The algal cell of claim 33, which is a part of a Nannochloropsis alga.