# Patent

## Title
Algal desaturases

## Abstract

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

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit and priority of U.S. Provisional Patent Application Ser. No. 61/480,353 filed on Apr. 28, 2011, titled “Desaturases,” which is hereby incorporated by reference.
The present application is related to U.S. Non-Provisional patent application Ser. No. 12/581,812 filed on Oct. 19, 2009, titled “Homologous Recombination in an Algal Nuclear Genome,” which is hereby incorporated by reference.
The present application is related to U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” which is hereby incorporated by reference.
The present application is related to U.S. Non-Provisional patent application Ser. No. 12/480,611 filed on Jun. 8, 2009, titled “Transformation of Algal Cells,” which is hereby incorporated by reference.


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 desaturases.
SUMMARY OF THE INVENTION
Isolated nucleotide sequences encoding polypeptides having desaturase activity, which utilize fatty acids as substrates.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a table of exemplary desaturases.
FIG. 2 illustrates the nucleotide sequence encoding desaturase 3 (SEQ ID NO:1).
FIG. 3 illustrates the nucleotide sequence encoding desaturase 5 (SEQ ID NO:2).
FIG. 4 illustrates the nucleotide sequence encoding desaturase 7 (SEQ ID NO:3).
FIG. 5 illustrates the nucleotide sequence encoding desaturase 9 (SEQ ID NO:4).
FIG. 6 illustrates the nucleotide sequence encoding desaturase 10 (SEQ ID NO:5).
FIG. 7 illustrates the amino acid sequence encoded by desaturase 3 (SEQ ID NO:6).
FIG. 8 illustrates the amino acid sequence encoded by desaturase 5 (SEQ ID NO:7).
FIG. 9 illustrates the amino acid sequence encoded by desaturase 7 (SEQ ID NO:8).
FIG. 10 illustrates the amino acid sequence encoded by desaturase 9 (SEQ ID NO:9).
FIG. 11 illustrates the amino acid sequence encoded by desaturase 10 (SEQ ID NO:10).



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. A fatty acid desaturase is an enzyme that removes two hydrogen atoms from a fatty acid, creating a carbon/carbon double bond. These desaturases are classified as either Delta desaturases, indicating that the double bond is created at a fixed position from the carboxyl group of a fatty acid (for example, a delta nine (“Δ9”) desaturase creates a double bond at the 9th position from the carboxyl end) or classified as Omega desaturases (for example, an omega three (“ω3”) desaturase, which creates the double bond between the third and fourth carbon from the methyl end of the fatty acid).
Provided herein are isolated nucleotide sequences encoding polypeptides having desaturase 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 desaturases, including exemplary desaturases which they designated as desaturase 3 (“desat3”), desaturase 5 (“desat5”), desaturase 7 (“desat7”), desaturase 9 (“desat9”), and desaturase 10 (“desat10”).
The inventors manipulated the activities of the above-specified exemplary desaturase genes by:
1. Overexpression of the subject desaturase gene with a strong promoter.
2. Promoter replacement or promoter insertion in front of the subject desaturase gene within the genome via homologous recombination.
3. Knock out of the subject desaturase gene via insertion of a transformation construct into the gene or replacement of a part of or the entire subject desaturase 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 desaturases for the purpose of modifying the contents of certain fatty acids within algal genus Nannochloropsis. 
FIG. 1 is a table of exemplary desaturases. The table includes the number of the respective desaturase, captioned “Desaturase No.”, the corresponding sequence identifier number, captioned “SEQ ID NO”, the type of desaturase, captioned “Desaturase Type”, the relative increase in desaturase gene regulation with respect to the presence or absence of Nitrogen, captioned “Times Up Regulated in the Presence/Absence of Nitrogen”, a first desaturase substrate, captioned “Substrate 1”, the resulting first product, captioned “Product 1”, a second desaturase substrate (if applicable), captioned “Substrate 2”, the resulting second product (if applicable), captioned “Product 2”, whether the desaturase gene was up-regulated or down-regulated in a particular mutant construct (with respect to the Substrate 1 to Product 1 reaction), captioned “Gene Regulation in Mutant Up/Down”, the observed increase or decrease of the corresponding substrate to product reaction by a mutant construct (as compared to a wild-type Nannochloropsis control cell), captioned “Substrate 1 to Product 1 Conversion” whether the desaturase gene was up-regulated or down-regulated in a particular mutant construct (with respect to the Substrate 2 to Product 2 reaction) (if applicable), captioned “Gene Regulation in Mutant Up/Down” and the observed increase or decrease of the corresponding substrate to product reaction by a mutant construct (as compared to a wild-type Nannochloropsis control cell), captioned “Substrate 2 to Product 2 Conversion” (if applicable).
As shown in FIG. 1, the inventors identified five exemplary desaturases, designated as desaturase 3 (“desat3”), desaturase 5 (“desat5”), desaturase 7 (“desat7”), desaturase 9 (“desat9”), and desaturase 10 (“desat10”). Each desaturase has a corresponding SEQ ID NO, reflecting a nucleotide sequence for the respective desaturase. The desaturase type (which indicates corresponding enzymatic activity) is also shown in FIG. 1. The column captioned “Times Up Regulated in the Presence/Absence of Nitrogen” reflects whole transcriptome comparisons of samples grown under Nitrogen sufficient (“N+”) or Nitrogen deficient conditions (“N−”). The columns captioned “Substrate 1 to Product 1 Conversion” and “Substrate 2 to Product 2 Conversion” (if applicable) show the observed increase or decrease of the corresponding substrate to product reactions by a mutant construct (as compared to a wild-type Nannochloropsis control cell).
Referring again to the table in FIG. 1, desaturase 3, a Delta 12 desaturase, is approximately 1.5 times up-regulated in the absence of Nitrogen. Desaturase 3 increases the conversion of Palmitolenic acid (16:2(n-7)) to 6,9,12-hexadecatrienoic acid (16:3(n-4)) by approximately 85% (when compared to a wild-type Nannochloropsis control cell). Desaturase 3 also increases the conversion of Oleic acid, cis-9-octadecenoic acid (18:1(n-9)) to Linoleic acid, all-cis-9, 12-octadecadienoic acid (18:2(n-6)) by approximately 50% (when compared to a wild-type Nannochloropsis control cell).
Desaturase 5, as shown in the table in FIG. 1, is also a Delta 12 desaturase. Desaturase 5 is not regulated in the presence or absence of Nitrogen. Desaturase 5 decreases the conversion of Oleic acid, cis-9-octadecenoic acid (18:1(n-9)) to 12-octadecadienoic acid (18:2(n-6)) by approximately 100% (when compared to a wild-type Nannochloropsis control cell).
Desaturase 7, shown in the table in FIG. 1, is an Omega 3 desaturase. Desaturase 7 is approximately 4.0 times up-regulated in the presence of Nitrogen. Overexpression of Desaturase 7 decreases the amount of Arachidonic acid, all-cis-5,8,11,14-eicosatetraenoic acid (20:4(n-6)) (“ARA”) by approximately 60% due to more conversion of ARA to Eicosapentaenoic acid, all-cis-5,8,11,14,17-eicosapentaenoic acid (20:5(n-3)) (“EPA”) (when compared to a wild-type Nannochloropsis control cell). In fact, the EPA to ARA ratio changes from about 7.5 to about 22, an approximate 200% increase. Desaturase 7 increases the conversion of Linoleic acid, all-cis-9, 12-octadecadienoic acid (18:2(n-6)) to a-Linolenic acid, all-cis-9,12,15-octadecatrienoic acid (18:3(n-3)) by approximately 650% (when compared to a wild-type Nannochloropsis control cell). In fact, the ratio of a-Linolenic acid, all-cis-9,12,15-octadecatrienoic acid (18:3(n-3)) to Linoleic acid, all-cis-9, 12-octadecadienoic acid (18:2(n-6)) changes from approximately 2.5 to approximately 15.8, an approximate 550% increase.
Desaturase 9, shown in the table in FIG. 1, is a Delta 9 desaturase. Desaturase 9 is approximately 6.6 times up-regulated in the absence of Nitrogen. Desaturase 9 increases the conversion of Palmitic acid, or hexadecanoic acid (16:0) to Palmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) by approximately 25%, while increasing the ratio of Palmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) to Palmitic acid, or hexadecanoic acid (16:0) from about 0.48 to 0.6, an approximate increase of 25%.
Desaturase 10, shown in the table in FIG. 1, is a Delta 9 desaturase. Desaturase 9 is approximately 5.0 times up-regulated in the absence of Nitrogen. Desaturase 9 increases the conversion of Palmitic acid, or hexadecanoic acid (16:0) to Palmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) by approximately 34%, while increasing the ratio of Palmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) to Palmitic acid, or hexadecanoic acid (16:0) from about 1.02 to 1.19, an approximate increase of 16%.
FIG. 2 illustrates the nucleotide sequence encoding desaturase 3 (SEQ ID NO:1).
The inventors found that desaturase 3 is slightly up-regulated under Nitrogen starvation. The inventors prepared a construct utilizing desaturase 3 that included a strong upstream promoter. This resulted in expression of elevated amounts of 16:3n4 and 18:2n6 fatty acids.
FIG. 3 illustrates the nucleotide sequence encoding desaturase 5 (SEQ ID NO:2).
The inventors found that desaturase 5 encodes a fatty acid desaturase with high homology to Delta 12 desaturases. The inventors also found that overexpression of this desaturase gene under the control of an inducible Urease promoter leads to higher expression levels of 18:1n9 fatty acids and poly unsaturated fatty acids (“PUFAs”) with 18 or more carbon atoms, when the constructs are grown under Nitrogen starvation conditions (please note: the promoter is induced under Nitrogen starvation).
In other experiments, the inventors determined that the desaturation step at the Delta 12 position is likely a major bottleneck for channeling carbon into the PUFA biosynthesis pathway. While 18:1n9 fatty acids are steadily increasing during Nitrogen starvation, 18:2n6 fatty acids (derived from the Delta 12 desaturation of said 18:1n9 fatty acids) are decreasing (on a percent basis of all fatty acids), as are all fatty acids in the pathway leading to the production of Eicosapentaenoic acid (“EPA”). The inventors concluded that the desaturase 5 gene increases carbon flux into the PUFA biosynthesis pathway during Nitrogen starvation if the desaturase 5 gene is over-expressed.
FIG. 4 illustrates the nucleotide sequence encoding desaturase 7 (SEQ ID NO:3).
The inventors prepared various constructs (promoter replacements, knock-outs, and over expression constructs) and found that down-regulation of the desaturase 7 gene results in a lower EPA/Arachidonic acid (“ARA”) ratio, i.e. less ARA is desaturated to EPA. The inventors observed in mutant constructs that lower desaturase 7 transcription is due to an exchange of the native promoter in the wild-type Nannochloropsis cells. The inventors observed that the mutant constructs had nearly double the levels of ARA with less levels of EPA, when compared to the wild-type Nannochloropsis control cells.
The inventors also observed that up-regulation of the desaturase 7 gene results in higher 18:3n3/18:2n6 and EPA/ARA ratios, i.e. more 18:2n6 is converted to 18:3n3, and more ARA is converted into EPA. Accordingly, the inventors observed that the EPA/ARA ratio was nearly doubled.
FIG. 5 illustrates the nucleotide sequence encoding desaturase 9 (SEQ ID NO:4).
FIG. 6 illustrates the nucleotide sequence encoding desaturase 10 (SEQ ID NO:5).
The inventors observed that both desaturase 9 and desaturase 10 appear to be Delta 9 desaturases acting primarily on 16:0 fatty acids or on 16:0 fatty acids attached to other compounds. Promotor exchange studies, in which the inventors exchanged the native wild-type promoter of Nannochloropsis against a strong promoter, revealed an up-regulation of said activity under Nitrogen deficient conditions. Thus, under Nitrogen starvation, a high percentage of fatty acids are channeled to the accumulation of 16:1n7 fatty acids through the action of the desaturase 9 gene, meaning less fatty acids are entering the PUFA pathway. The inventors also replaced the promoters of the desaturase 9 genes with promoters of moderate strength and which are putatively not regulated when cells enter Nitrogen starvation, with the goal to avoid carbon flux into the biosynthesis of 16:1n7 and 18:1n7 fatty acids and to increase carbon flux into the PUFA biosynthesis pathway during starvation. The inventors found that 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 desaturase gene may 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 desaturase gene, presumably by modifying the respective chromatin arrangement around the desaturase gene, thus leading to a lower transcription level. Also, the introduction of point mutations into the desaturase gene when inserting another promoter in front of the desaturase gene via the homologous recombination flanks may lead to an altered activity of the respective gene products.
The inventors also observed an increase of 16:1n7 lipids in a selected desaturase 10 over expression mutant under nitrogen sufficient conditions, clearly demonstrating that there is an approximately 34% increase in 16:1n7 fatty acids in this mutant when compared to the wild-type Nannochloropsis cells during the same experiment.
FIG. 7 illustrates the amino acid sequence encoded by desaturase 3 (SEQ ID NO:6).
FIG. 8 illustrates the amino acid sequence encoded by desaturase 5 (SEQ ID NO:7).
FIG. 9 illustrates the amino acid sequence encoded by desaturase 7 (SEQ ID NO:8).
FIG. 10 illustrates the amino acid sequence encoded by desaturase 9 (SEQ ID NO:9).
FIG. 11 illustrates the amino acid sequence encoded by desaturase 10 (SEQ ID NO:10).
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. An isolated nucleotide sequence encoding a polypeptide having desaturase activity, the nucleotide sequence having at least 95% sequence identity to SEQ ID NO:3.

2. The isolated nucleotide sequence of claim 1 wherein the sequence encodes a functionally active desaturase which utilizes a fatty acid as a substrate.

3. The isolated nucleotide sequence of claim 2, wherein the functionally active desaturase comprises amino acids having the sequence set forth as SEQ ID NO:8 or a sequence at least 95% identical thereto.

4. An isolated nucleotide sequence encoding a polypeptide having desaturase activity, the nucleotide sequence having at least 95% sequence identity to SEQ ID NO:4.

5. The isolated nucleotide sequence of claim 4 wherein the sequence encodes a functionally active desaturase which utilizes a fatty acid as a substrate.

6. The isolated nucleotide sequence of claim 5, wherein the functionally active desaturase comprises amino acids having the sequence set forth as SEQ ID NO:9 or a sequence at least 95% identical thereto.

7. An isolated nucleotide sequence encoding a polypeptide having desaturase activity, the nucleotide sequence having at least 95% sequence identity to SEQ ID NO:5.

8. The isolated nucleotide sequence of claim 7 wherein the sequence encodes a functionally active desaturase which utilizes a fatty acid as a substrate.

9. The isolated nucleotide sequence of claim 8, wherein the functionally active desaturase comprises amino acids having the sequence set forth as SEQ ID NO:10 or a sequence at least 95% identical thereto.

10. The isolated nucleotide sequence of claim 1 wherein the sequence is derived from algae.

11. The isolated nucleotide sequence of claim 10 wherein the algae is of genus Nannochloropsis.