HK1177464B - Polyunsaturated fatty acid synthase nucleic acid molecules and polypeptides, compositions, and methods of making and uses thereof - Google Patents

Description

Polyunsaturated fatty acid synthase nucleic acid molecules and polypeptides, compositions, preparation methods, and uses thereof

Background of the Invention

Field of the Invention

The present invention relates to isolated nucleic acid molecules and polypeptides encoding polyunsaturated fatty acid (PUFA) synthases involved in PUFA production, wherein the PUFAs include PUFAs rich in docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or a combination thereof. The present invention also relates to vectors and host cells containing the nucleic acid molecules and polypeptides encoded by the nucleic acid molecules, compositions containing the nucleic acid molecules or polypeptides, and methods for preparing and using the nucleic acid molecules or polypeptides.

Background of the Invention

Thraustochytrium is a microorganism of the order Thraustochytriales, including the genera Thraustochytrium and Schizochytrium, and is considered an alternative source of PUFAs. See, for example, U.S. Patent No. 5,130,242. Recently, polyketide synthase (PKS)-like systems have been discovered in marine bacteria and thraustochytrium that can synthesize polyunsaturated fatty acids (PUFAs) from acetyl-CoA and malonyl-CoA. These PKS synthase-like systems are also referred to herein as PUFA synthase systems. U.S. Patent No. 6,140,486 describes PUFA systems in the marine bacteria Shewanella and Vibrio marinus. U.S. Patent No. 6,566,583 describes a PUFA synthase system in the thraustochytrium genus Schizochytrium. U.S. Patent 7,247,461 describes a PUFA synthase system in thraustochytrium of the genera Schizochytrium and Thraustochytrium. U.S. Patent 7,211,418 describes a PUFA synthase system in thraustochytrium of the genus Thraustochytrium and uses the system to produce eicosapentaenoic acid (C20:5, Ω3) (EPA) and other PUFAs. U.S. Patent 7,217,856 describes PUFA synthases in Shewanella olleyana and Shewanella japonica. WO 2005/097982 describes a PUFA synthase in strain SAM2179. U.S. Patents 7,208,590 and 7,368,552 describe PUFA synthase genes and proteins from Thraustochytrium aureum.

The PKS systems classically described in the literature are of one of three basic types, usually referred to as Type I (modular or iterative), Type II, and Type III. Type I modular PKS systems are also referred to as "modular" PKS systems, and Type I iterative PKS systems are also referred to as "Type I" PKS systems. Type II systems are characterized by separable proteins, each of which performs a different enzymatic reaction. The enzymes work together to produce the end product, and each enzyme in the system participates several times in the production of the end product. This type of system operates in a similar way to the fatty acid synthase (FAS) system in plants and bacteria. Type I iterative PKS systems are similar to Type II systems in that enzymes are used to produce the end product in an iterative manner. The difference between Type I iterative systems and Type II systems is that their enzymatic activity is not associated with separable proteins, but is present in the structural domain of larger proteins. This system is similar to the Type I FAS system in animals and fungi.

Unlike Type II systems, each enzyme domain in a Type I modular PKS system is used only once in the process of producing the final product. The domains are present in very large proteins, and the product of each reaction is passed to another domain of the PKS protein.

The recently discovered type III system belongs to the chalcone synthase family of plant condensing enzymes. Type III PKSs differ from type I and type II PKS systems in that they typically utilize free CoA substrates to produce heterocyclic end products through iterative condensation reactions.

In the conventional or standard pathway for PUFA synthesis, medium-chain saturated fatty acids (products of the fatty acid synthase (FAS) system) are modified through a series of elongation and desaturation reactions. The substrates for the elongation reactions are fatty acyl-CoA (the fatty acid chain to be elongated) and malonyl-CoA (the source of the two carbons added in each elongation reaction). The product of the elongase reaction is a fatty acyl-CoA with two carbons added to the linear chain. Desaturases create cis-double bonds in existing fatty acid chains by abstracting two hydrogens in an oxygen-dependent reaction. The substrates for desaturases are acyl-CoA (in some animals) or fatty acids esterified to the glycerol backbone of phospholipids (such as phosphatidylcholine).

Fatty acids are classified based on their carbon chain length and saturation characteristics. Depending on the number of carbon atoms in the chain, they can be classified as short-chain, medium-chain, or long-chain fatty acids. When there are no double bonds between the carbon atoms, they are called saturated fatty acids, while when double bonds are present, they are called unsaturated fatty acids. Unsaturated long-chain fatty acids are monounsaturated when only one double bond is present, and polyunsaturated when more than one double bond is present.

PUFAs are classified according to the position of the first double bond at the methyl end of the fatty acid: ω-3 (n-3) fatty acids have their first double bond at the third carbon, while ω-6 (n-6) fatty acids have their first double bond at the sixth carbon. For example, docosahexaenoic acid ("DHA") is a 22-carbon ω-3 PUFA with six double bonds, often designated "22:6 n-3." Other ω-3 PUFAs include eicosapentaenoic acid ("EPA"), designated "20:5 n-3," and ω-3 docosapentaenoic acid ("DPA n-3"), designated "22:5 n-3." DHA and EPA are considered "essential" fatty acids. ω-6 PUFAs include arachidonic acid ("ARA"), designated "20:4 n-6," and ω-6 docosapentaenoic acid ("DPA n-6"), designated "22:5 n-6."

Because they appear on cell membranes, ω-3 fatty acids are important biological molecules that affect the physiological functions of cells. They can regulate the production of bioactive compounds and gene expression, and serve as substrates for biosynthesis. Roche, H.M., Proc. Nutr. Soc. 58: 397-401 (1999). For example, DHA accounts for about 15%-20% of the lipids in the human cerebral cortex and 30%-60% of the retinal lipids. It is enriched in testicles and sperm and is an important component of breast milk. Bergé, J.P., and Barnathan, G. Adv. Biochem. Eng. Biotechnol. 96: 49-125 (2005). DHA accounts for up to 97% of the ω-3 fatty acids in the brain and up to 93% of the ω-3 fatty acids in the retina. Moreover, DHA is necessary for fetal and infant development, as well as for the maintenance of cognitive function in adults. Same as above. Because ω-3 fatty acids are not synthesized de novo in the human body, these fatty acids must be derived from nutritional ingredients.

Flaxseed oil and fish oil are recognized as good dietary sources of omega-3 fatty acids. Flaxseed oil does not contain EPA, DHA, DPA, or ARA, but does contain linolenic acid (C18:3 n-3), a building block for the body to make EPA. However, there is evidence that its metabolic conversion rate is slow and variable, particularly in patients with compromised health. The type and amount of fatty acid composition in fish oils varies significantly, depending on the species and its diet. For example, aquacultured fish have lower levels of omega-3 fatty acids than wild fish. Furthermore, fish oils carry the risk of containing environmental contaminants, stability issues, and a fishy odor or taste.

Oils produced by thraustochytrids often have a simpler polyunsaturated fatty acid profile than corresponding fish oils or bacterial oils. Lewis, T.E., Mar. Biotechnol. 1:580-587 (1999). It has been reported that strains of thraustochytrids produce a high proportion of ω-3 fatty acids in the total fatty acids produced by the organism. U.S. Patent 5,130,242; Huang, J. et al., J. Am. Oil. Chem. Soc. 78:605-610 (2001); Huang, J. et al., Mar. Biotechnol. 5:450-457 (2003). However, isolated thraustochytrids vary in the types and amounts of PUFAs produced, and therefore some previously described strains may have undesirable PUFA profiles.

PUFAs are produced in oilseed crops by modifying endogenously produced fatty acids. Plants genetically modified with different individual genes for fatty acid elongases and desaturases produce leaves or seeds that contain detectable levels of PUFAs such as EPA, but also contain significant levels of mixed short-chain and less unsaturated PUFAs (Qi et al., Nature Biotech. 22:739 (2004); PCT Publication No. WO 04/071467; Abbadi et al., Plant Cell 16:1 (2004); Napier and Sayanova, Proc. Nutrition Society 64:387-393 (2005); Robert et al., Functional Plant Biology 32:473-479 (2005); and U.S. Application Publication No. 2004/0172682).

Therefore, there is a need for isolating nucleic acid molecules and polypeptides associated with a desired PUFA profile, and for methods of producing a desired PUFA profile using such nucleic acid molecules and polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 1, wherein the polynucleotide sequence encodes a polypeptide comprising a polyunsaturated fatty acid (PUFA) synthase activity selected from the group consisting of β-ketoacyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity, ketoreductase (KR) activity, β-hydroxyacyl-ACP dehydratase (DH) activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 7, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 9, wherein the polynucleotide sequence encodes a polypeptide comprising MAT activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 13, 15, 17, 19, 21, or 23, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity;

(e) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 11, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity;

(f) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 25, wherein the polynucleotide sequence encodes a polypeptide comprising KR activity; and

and (g) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 27, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity. In some embodiments, the polynucleotide sequence is at least 90% or at least 95% identical to SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 8, and the polypeptide comprises KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10, and the polypeptide comprises MAT activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 14, 16, 18, 20, or 24, and the polypeptide comprises ACP activity;

(e) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12, and the polypeptide comprises ACP activity;

(f) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 26, and the polypeptide comprises KR activity;

and (g) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 28, and the polypeptide comprises DH activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NO: 2, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 3, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, chain length factor (CLF) activity, acyltransferase (AT) activity, enoyl-ACP reductase (ER) activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 29, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 31, wherein the polynucleotide sequence encodes a polypeptide comprising CLF activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 33, wherein the polynucleotide sequence encodes a polypeptide comprising AT activity; and

and (e) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 35, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity. In some embodiments, the polynucleotide sequence is at least 90% or 95% identical to SEQ ID NOs: 3, 29, 31, 33, and 35, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NOs: 3, 29, 31, 33, and 35, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 30, and the polypeptide comprises KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 32, and the polypeptide comprises CLF activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 34, and the polypeptide comprises AT activity;

and (e) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 36, and the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 4, 30, 32, 34, and 36, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 4, 30, 32, 34, and 36, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 5, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 37, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 39, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity;

and (d) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 41, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity; in some embodiments, the polynucleotide sequence is at least 90% or 95% identical to SEQ ID NOs: 5, 37, 39, and 41, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequences of SEQ ID NOs: 5, 37, 39, and 41, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 6, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 38, and the polypeptide comprises DH activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 40, and the polypeptide comprises DH activity;

and (d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 42, and the polypeptide comprises ER activity; in some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 6, 38, 40, and 42, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 6, 38, 40, and 42, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 68 or 120, wherein the polynucleotide encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 74, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 76, wherein the polynucleotide sequence encodes a polypeptide comprising MAT activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity;

(e) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 78, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity;

(f) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 100, wherein the polynucleotide sequence encodes a polypeptide comprising KR activity; and

and (g) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 118, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity. In some embodiments, the polynucleotide sequence is at least 90% or 95% identical to SEQ ID NOs: 68, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 118, and 120, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NOs: 68, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 118, and 120.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 69, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 75, and the polypeptide comprises KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 77, and the polypeptide comprises MAT activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 81, 83, 87, 89, 91, 93, 95, 97, or 99, and the polypeptide comprises ACP activity;

(e) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 79, and the polypeptide comprises ACP activity;

(f) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 101, and the polypeptide comprises KR activity;

and (g) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 119, and the polypeptide comprises DH activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NO: 69, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, and 119, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 69, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, and 119.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 70 or SEQ ID NO: 121, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, chain length factor (CLF) activity, acyltransferase (AT) activity, enoyl reductase (ER) activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 102, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 104, wherein the polynucleotide sequence encodes a polypeptide comprising CLF activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 106, wherein the polynucleotide sequence encodes a polypeptide comprising AT activity; and

and (e) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 108, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity. In some embodiments, the polynucleotide sequence is at least 90% or 95% identical to SEQ ID NOs: 70, 102, 104, 106, 108, and 121, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NOs: 70, 102, 104, 106, 108, and 121, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 71, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 103, and the polypeptide comprises KS activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 105, and the polypeptide comprises CLF activity;

(d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 107, and the polypeptide comprises AT activity;

and (e) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 109, and the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NO: 71, 103, 105, 107, and 109, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 71, 103, 105, 107, and 109, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 72 or 122, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 110, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 112, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity;

and (d) a nucleic acid molecule comprising a polynucleotide sequence at least 80% identical to SEQ ID NO: 114, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity. In some embodiments, the polynucleotide sequence is at least 90% or 95% identical to SEQ ID NOs: 72, 110, 112, 114, and 122, respectively. In some embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NOs: 72, 110, 112, 114, and 122, respectively.

The present invention relates to an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 73, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 111, and the polypeptide comprises DH activity;

(c) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 113, and the polypeptide comprises DH activity;

and (d) a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 115, and the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NO: 73, 111, 113, and 115, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 73, 111, 113, and 115, respectively.

The present invention relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, CLF activity, AT activity, ER activity, DH activity, and combinations thereof, wherein the polynucleotide hybridizes under stringent conditions to the complement of any of the above polynucleotide sequences.

The present invention relates to an isolated nucleic acid molecule comprising a polynucleotide sequence that is fully complementary to any of the polynucleotide sequences described above.

The present invention relates to a recombinant nucleic acid molecule comprising any of the above nucleic acid molecules and combinations thereof and a transcription control sequence. In some embodiments, the recombinant nucleic acid molecule is a recombinant vector.

The present invention relates to host cells expressing any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant nucleic acid molecules, and combinations thereof. In some embodiments, the host cell is selected from plant cells, microbial cells, and animal cells. In some embodiments, the microbial cell is a bacterium. In some embodiments, the bacterium is Escherichia coli (E. coli). In some embodiments, the bacterium is a marine bacterium. In some embodiments, the microbial cell is a thraustochytrium. In some embodiments, the thraustochytrium is a Schizochytrium. In some embodiments, the thraustochytrium is a Thraustochytrium. In some embodiments, the thraustochytrium is an Ulkenia.

The present invention relates to a method for producing at least one PUFA, comprising: expressing a PUFA synthase gene in a host cell under conditions effective to produce the PUFA, wherein the PUFA synthase gene comprises any of the above-described isolated nucleic acid molecules, any of the above-described recombinant nucleic acid molecules, or a combination thereof, and producing the at least one PUFA. In one aspect of this embodiment, the host cell is selected from a plant cell, an isolated animal cell, and a microbial cell. In another aspect of this embodiment, the at least one PUFA comprises docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

The present invention relates to a method for producing lipids enriched in DHA, EPA, or a combination thereof, comprising: expressing a PUFA synthase gene in a host cell under conditions effective to produce the lipids, wherein the PUFA synthase gene comprises any of the above-described isolated nucleic acid molecules, any of the above-described recombinant nucleic acid molecules, or a combination thereof, and producing lipids enriched in DHA, EPA, or a combination thereof. The present invention also relates to a method for producing a recombinant vector, comprising inserting any of the above-described isolated nucleic acid molecules into a vector.

The present invention relates to a method for producing a recombinant host cell, comprising introducing the above-mentioned recombinant vector into a host cell. In some embodiments, the host cell is selected from plant cells, isolated animal cells and microbial cells.

The present invention relates to isolated polypeptides encoded by any of the above-mentioned polynucleotide sequences.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 8, wherein the polypeptide comprises KS activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 10, wherein the polypeptide comprises MAT activity;

(d) a polypeptide comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 14, 16, 18, 20, or 24, wherein the polypeptide comprises ACP activity;

(e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 12, wherein the polypeptide comprises ACP activity;

(f) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 26, wherein the polypeptide comprises KR activity; and

and (g) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 28, wherein the polypeptide comprises DH activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28, respectively.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 4, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 30, wherein the polypeptide comprises KS activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 32, wherein the polypeptide comprises CLF activity;

(d) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 34, wherein the polypeptide comprises AT activity; and

and (e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 36, wherein the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 4, 30, 32, 34, and 36, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 4, 30, 32, 34, and 36, respectively.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 6, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 38, wherein the polypeptide comprises DH activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 40, wherein the polypeptide comprises DH activity;

and (e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 42, wherein the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 6, 38, 40, and 42, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 6, 38, 40, and 42, respectively.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 69, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 75, wherein the polypeptide comprises KS activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 77, wherein the polypeptide comprises MAT activity;

(d) a polypeptide comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99, wherein the polypeptide comprises ACP activity;

(e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 79, wherein the polypeptide comprises ACP activity;

(f) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 101, wherein the polypeptide comprises KR activity; and

and (g) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 119, wherein the polypeptide comprises DH activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 69, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, and 119, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 69, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, and 119.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 71, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 103, wherein the polypeptide comprises KS activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 105, wherein the polypeptide comprises CLF activity;

(d) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 107, wherein the polypeptide comprises AT activity; and

And (e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 109, wherein the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 71, 103, 105, 107, and 109, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 71, 103, 105, 107, and 109, respectively.

The present invention relates to an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 73, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof;

(b) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 111, wherein the polypeptide comprises DH activity;

(c) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 113, wherein the polypeptide comprises DH activity;

and (e) a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 115, wherein the polypeptide comprises ER activity. In some embodiments, the amino acid sequence is at least 90% or 95% identical to SEQ ID NOs: 73, 111, 113, and 115, respectively. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NOs: 73, 111, 113, and 115, respectively.

In some embodiments, any of the isolated polypeptides of the present invention can be a fusion polypeptide.

The present invention relates to a composition comprising any of the above-mentioned polypeptides and a biologically acceptable carrier.

The present invention relates to a method for increasing the production of DHA, EPA or a combination thereof in an organism having PUFA synthase activity, comprising: expressing any of the above-described isolated nucleic acid molecules, any of the above-described recombinant nucleic acid molecules, or a combination thereof in the organism under conditions effective to produce DHA, EPA or a combination thereof, wherein the PUFA synthase activity replaces an inactivated or deleted activity, introduces a new activity, or enhances an existing activity in the organism, wherein the production of DHA, EPA or a combination thereof in the organism is increased.

The present invention relates to a method for isolating lipids from a host cell, comprising (a) expressing a PUFA synthase gene in the host cell under conditions effective to produce the lipids, wherein the PUFA synthase gene comprises any of the above-described isolated nucleic acid molecules, any of the above-described recombinant nucleic acid molecules, or a combination thereof, and (b) isolating the lipids from the host cell. In some embodiments, the host cell is selected from a plant cell, an isolated animal cell, and a microbial cell. In some embodiments, the lipids comprise DHA, EPA, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the gene structure of the Schizochytrium sp. ATCC PTA-9695 PUFA synthase of the present invention.

FIG2 shows the gene structure of the Thraustochytrium sp. ATCC PTA-10212 PUFA synthase of the present invention.

3 shows the domain structures of Schizochytrium sp. ATCC PTA-9695 and Thraustochytrium sp. ATCC PTA-10212 PUFA synthases of the present invention, as well as synthases from Schizochytrium sp. ATCC 20888, Thraustochytrium sp. ATCC 20892, Thraustochytrium aureum, and SAM2179.

Figure 4 shows an alignment of the Schizochytrium sp. ATCC PTA-9695 Pfa1p amino acid sequence (SEQ ID NO: 2) and the Thraustochytrium sp. ATCC PTA-10212 Pfa1p amino acid sequence (SEQ ID NO: 69) of the present invention with the OrfA sequences from Schizochytrium sp. ATCC 20888 (SEQ ID NO: 54) and Thraustochytrium sp. ATCC 20892 (SEQ ID NO: 56), and the ORF A sequence from Thraustochytrium aureum (SEQ ID NO: 55).

Figure 5 shows an alignment of the Schizochytrium sp. ATCC PTA-9695 Pfa2p amino acid sequence (SEQ ID NO: 4) and the Thraustochytrium sp. ATCC PTA-10212 Pfa2p amino acid sequence (SEQ ID NO: 71) of the present invention with the OrfB sequences from Schizochytrium sp. ATCC 20888 (SEQ ID NO: 57) and Thraustochytrium sp. ATCC 20892 (SEQ ID NO: 58), and the ORF B sequence from Thraustochytrium aureum (SEQ ID NO: 59).

Figure 6 shows an alignment of the Schizochytrium sp. ATCC PTA-9695 Pfa3p amino acid sequence (SEQ ID NO: 6) and the Thraustochytrium sp. ATCC PTA-10212 Pfa3p amino acid sequence (SEQ ID NO: 73) of the present invention with the ORF C sequence from Schizochytrium sp. ATCC 20888 (SEQ ID NO: 61) and Thraustochytrium sp. ATCC 20892 (SEQ ID NO: 60).

FIG. 7 shows the polynucleotide sequence of Schizochytrium sp. ATCC PTA-9695 PFA1 (SEQ ID NO: 1).

FIG8 shows the amino acid sequence of Schizochytrium sp. ATCC PTA-9695 Pfa1p (SEQ ID NO: 2).

FIG. 9 shows the polynucleotide sequence of Schizochytrium sp. ATCC PTA-9695 PFA2 (SEQ ID NO: 3).

FIG. 10 shows the amino acid sequence of Schizochytrium sp. ATCC PTA-9695 Pfa2p (SEQ ID NO: 4).

FIG. 11 shows the polynucleotide sequence of Schizochytrium sp. ATCC PTA-9695 PFA3 (SEQ ID NO: 5).

FIG. 12 shows the amino acid sequence of Schizochytrium sp. ATCC PTA-9695 Pfa3p (SEQ ID NO: 6).

FIG. 13 shows the polynucleotide sequence of Schizochytrium sp. ATCC PTA-10212 PFA1 (SEQ ID NO: 68).

FIG. 14 shows the polynucleotide sequence of Thraustochytrium sp. ATCC PTA-10212 PFA1 codon-optimized for expression in Schizochytrium (SEQ ID NO: 120).

FIG. 15 shows the amino acid sequence of Thraustochytrium sp. ATCC PTA-10212 Pfa1p (SEQ ID NO: 69).

FIG. 16 shows the polynucleotide sequence of Thraustochytrium sp. ATCC PTA-10212 PFA2 (SEQ ID NO: 70).

FIG. 17 shows the polynucleotide sequence of Thraustochytrium sp. ATCC PTA-10212 PFA2 (SEQ ID NO: 121) codon-optimized for expression in Schizochytrium.

FIG. 18 shows the amino acid sequence of Thraustochytrium sp. ATCC PTA-10212 Pfa2p (SEQ ID NO: 71).

FIG. 19 shows the polynucleotide sequence of Thraustochytrium sp. ATCC PTA-10212 PFA3 (SEQ ID NO: 72).

FIG. 20 shows the polynucleotide sequence of Thraustochytrium sp. ATCC PTA-10212 PFA3 (SEQ ID NO: 122) codon-optimized for expression in Schizochytrium.

FIG. 21 shows the amino acid sequence of Thraustochytrium sp. ATCC PTA-10212 Pfa3p (SEQ ID NO: 73).

Figure 22 shows a codon usage table for Schizochytrium.

Detailed Description of the Invention

The present invention relates to isolated nucleic acid molecules and polypeptides that are involved in the production of polyunsaturated fatty acid (PUFA) synthases, including PUFAs rich in docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and combinations thereof. The present invention also relates to vectors and host cells containing the nucleic acid molecules and polypeptides encoded by the nucleic acid molecules, compositions containing the nucleic acid molecules or polypeptides, and methods for preparing and using the compositions.

PUFA synthase

As used herein, the term "PUFA synthase" refers to an enzyme involved in the production of polyunsaturated fatty acids. See, for example, Metz et al., Science 293:290-293 (2001).

The present invention relates, in part, to three PUFA synthase subunits designated Pfa1p (SEQ ID NO:2 or SEQ ID NO:69), Pfa2p (SEQ ID NO:4 or SEQ ID NO:71), and Pfa3p (SEQ ID NO:6 or SEQ ID NO:73), as well as genes encoding subunits designated PFA1 (SEQ ID NO:1, SEQ ID NO:68, or SEQ ID NO:120), PFA2 (SEQ ID NO:3, SEQ ID NO:70, or SEQ ID NO:121), and PFA3 (SEQ ID NO:5, SEQ ID NO:72, or SEQ ID NO:122). See Figures 1-3 and 7-21. PUFA synthases from other thraustochytrids are designated ORF 1, ORF 2, and ORF 3, or OrfA, OrfB, and OrfC, respectively. See U.S. Patent Nos. 7,247,461 and 7,256,022 for the orfA, orfB, and orfC genes and corresponding OrfA, OrfB, and OrfC proteins of Schizochytrium sp. (ATCC 20888) and Thraustochytrium sp. (ATCC 20892), and U.S. Patent No. 7,368,552 for the ORFA, ORFB, and ORFC genes and proteins of Thraustochytrium aureum (ATCC 34304). See WO/2005/097982 for the SAM2179 strain for the ORF1, ORF2, and ORF3 genes and proteins.

Nucleic acid molecules

The present invention relates to isolated nucleic acid molecules comprising polynucleotide sequences of PUFA synthase genes, and domains derived from the isolated microorganism, which is the subject of co-pending U.S. application Ser. No. 12/407,687, filed on March 19, 2009, which is incorporated by reference in its entirety. The microorganism was deposited under the Budapest Treaty with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, on January 7, 2009, under ATCC Accession No. PTA-9695, also known as Schizochytrium sp. ATCC PTA-9695. Expression of these genes results in a unique fatty acid profile characterized in part by high levels of omega-3 fatty acids, particularly DHA.

The present invention relates to isolated nucleic acid molecules comprising polynucleotide sequences of PUFA synthase genes, and domains derived from the isolated microorganism, which is the subject of U.S. Application No. 61/296,456, filed January 19, 2010, which is incorporated by reference in its entirety. The microorganism was deposited with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, under the Budapest Treaty, on July 14, 2009, under ATCC Accession No. PTA-10212, also known as Thraustochytrium sp. ATCC PTA-10212. Expression of these genes results in a unique fatty acid profile characterized, in part, by high levels of omega-3 fatty acids, particularly high levels of DHA, EPA, and combinations thereof.

As used herein, "polynucleotide" may comprise conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as the amide bonds in peptide nucleic acids (PNA)). A polynucleotide may comprise the nucleotide sequence of a full-length cDNA sequence, including untranslated 5' and 3' sequences, coding sequences, as well as fragments, epitopes, domains, and nucleic acid sequence variants. A polynucleotide may be composed of any polyribonucleotide or polydeoxyribonucleotide, and may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed of single-stranded or double-stranded DNA, single-stranded or double-stranded region mixture DNA, single-stranded or double-stranded RNA, single-stranded or double-stranded region mixture RNA, a hybrid molecule comprising a single-stranded or more typically double-stranded or single-stranded and double-stranded region mixture of DNA and RNA. In addition, a polynucleotide may be composed of a triple-stranded region comprising RNA or DNA or RNA and DNA. A polynucleotide may comprise ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules") or any phosphate analogs thereof, such as phosphorothioates and thioesters. A polynucleotide may also comprise one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, trityl bases and unusual bases such as inosine. DNA and RNA can be modified in a variety of ways; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms. The term nucleic acid molecule refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term encompasses double-stranded DNA, particularly double-stranded DNA in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein in the general convention that the sequence is given only in the 5' to 3' direction along the non-transcribed DNA strand (ie, the strand with mRNA homologous sequence).

The term "isolated" nucleic acid molecule refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. Other examples of isolated nucleic acid molecules include nucleic acid molecules containing recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially completely) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. According to the present invention, isolated nucleic acid molecules also include synthetically produced such molecules. In addition, the nucleic acid molecule or polynucleotide may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

"Gene" refers to an assembly of nucleotides that encodes a polypeptide, including cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid segment that expresses a specific protein, including intervening sequences (introns) between coding segments (exons), as well as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene that occurs in nature with its own regulatory sequences.

The present invention relates to isolated nucleic acid molecules comprising a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence of Schizochytrium ATCC PTA-9695 PFA1 (SEQ ID NO: 1), Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3), Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5), Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 68 or SEQ ID NO: 120), Thraustochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 70 or SEQ ID NO: 121), Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 72 or SEQ ID NO: 122), and combinations thereof, wherein the polynucleotide encodes a polypeptide comprising one or more PUFA synthase activities.

PUFA synthase activity is associated with one or more domains within each synthase polypeptide, wherein the domains can be identified by homology to known motifs through conserved structural or functional motifs, or based on specific biochemical activity. See, for example, U.S. Patent No. 7,217,856, which is incorporated herein by reference in its entirety. Examples of PUFA synthase domains include: a β-ketoacyl ACP synthase (KS) domain, a malonyl-CoA:ACP acyltransferase (MAT) domain, an acyl carrier protein (ACP) domain, a ketoreductase (KR) domain, and a β-hydroxyacyl-ACP dehydratase (DH) domain in Pfa1p; a KS domain, a chain length factor (CLF) domain, an acyltransferase (AT) domain, and an enoyl-ACP reductase (ER) domain in Pfa2p; and a DH domain and an ER domain in Pfa3p.

Polypeptides or polypeptide domains having β-ketoacyl-ACP synthase (KS) biological activity (function) have previously been shown to be capable of carrying out the initial step of the fatty acid elongation reaction cycle. The term "β-ketoacyl-ACP synthase" is used interchangeably with the terms "3-ketoacyl-ACP synthase,""β-ketoacyl-ACPsynthase," and "ketoacyl-ACP synthase." In other systems, the acyl group for elongation is linked to a cysteine residue via a thioester bond in the KS active site, and the acyl-KS condenses with malonyl-ACP to form ketoacyl-ACP, _CO2 , and unbonded ("free") KS. In such systems, KS exhibits enhanced substrate specificity compared to other polypeptides in the reaction cycle. Polypeptides (or domains of polypeptides) can be readily identified as belonging to the KS family by homology to known KS sequences.

Polypeptides or polypeptide domains having malonyl-CoA:ACP acyltransferase (MAT) activity are shown to be able to transfer a malonyl moiety from a malonyl-CoA moiety to an ACP. The terms "malonyl-CoA:ACP acyltransferase" and "malonyl acyltransferase" are used interchangeably. In addition to the active site motif (GxSxG), MATs also possess an extended motif (R and Q amino acids at key positions). Polypeptides (or domains of polypeptides) belonging to the MAT family can be readily identified by homology to known MAT sequences or by the structure of the extended motif.

Polypeptides or polypeptide domains with acyl carrier protein (ACP) activity are shown to function as carriers for the growth of fatty acyl chains via thioester linkage to a covalently bound cofactor. ACPs are typically approximately 80-100 amino acids in length and are converted from an inactive apoprotein form to an active holoprotein form by transferring the phosphopantetheinyl moiety of CoA to a highly conserved serine residue in the ACP. The acyl group is linked to the ACP at the free end of the phosphopantetheinyl moiety via a thioester linkage. The presence of various active site motifs (LGIDS*) is also considered a characteristic of ACPs. The functionality of the active site serine (S*) has been demonstrated in bacterial PUFA synthases (Jiang et al., J. Am. Chem. Soc. 130:6336-7 (2008)). Polypeptides (or polypeptide domains) belonging to the ACP family can be readily identified using radioactive pantethein labeling or by sequence homology to known ACPs.

A polypeptide or polypeptide domain having dehydratase or dehydratase (DH) activity exhibits the ability to catalyze a dehydration reaction. Reference to DH activity generally refers to the biological activity of a FabA-like β-hydroxyacyl-ACP dehydratase. The biological activity of a FabA-like β-hydroxyacyl-ACP dehydratase removes HOH from β-ketoacyl-ACP, initially creating a trans double bond in the carbon chain. The term "FabA-like β-hydroxyacyl-ACP dehydratase" is used interchangeably with the terms "FabA-like β-hydroxyacyl-ACP dehydratase," "β-hydroxyacyl-ACP dehydratase," and "dehydratase." The DH domain of the PUFA synthase system is homologous to the bacterial DH enzymes associated with the FAS system, but not to the DH domains of other PKS systems. See, for example, U.S. Patent No. 7,217,856, which is incorporated herein by reference in its entirety. A subset of bacterial DHs, FabA-like DHs, possess cis-trans isomerase activity (Heath et al., J. Biol. Chem., 271, 27795 (1996)). Based on homology to FabA-like DH proteins, one or all PUFA synthase system DH domains can insert a cis double bond into the PUFA synthase product. The polypeptide or domain may also possess non-FabA-like DH activity, or non-FabA-like β-hydroxyacyl-ACP dehydratase (DH) activity. More specifically, a conserved active site motif of approximately 13 amino acids has been identified in the PUFA synthase DH domain: LxxHxxxGxxxxP (the L position in the motif can also be I). See, for example, U.S. Patent No. 7,217,856 and Donadio S, Katz L., Gene 111(1): 51-60 (1992), each of which is incorporated by reference in its entirety. This conserved motif can be found in similar regions in all known PUFA synthase sequences and may be associated with non-FabA-like dehydration.

Polypeptides or polypeptide domains having β-ketoacyl-ACP reductase (KR) activity are shown to catalyze the pyridine-nucleotide-dependent reduction of the 3-ketoacyl form of ACP. The term "β-ketoacyl-ACP reductase" is used interchangeably with "ketoreductase," "3-ketoacyl-ACP reductase," and "ketoacyl ACP reductase." KR function has been determined in other systems to include the first reduction step in the elongation cycle of de novo fatty acid biosynthesis. Polypeptides (or polypeptide domains) belonging to the KR family can be readily identified by sequence homology to known PUFA synthase KRs.

A polypeptide or polypeptide domain having chain length factor (CLF) activity has been previously defined as having one or more of the following activities or characteristics: (1) determining the number of elongation cycles, and therefore the chain length of the final product,

(2) Homologous to KS, but lacking the cysteine in the KS active site,

(3) Can heterodimerize with KS,

(4) can provide the initial acyl group for extension,

Alternatively, (5) the malonate can be decarboxylated (forming malonyl-ACP), thereby forming an acetate group that can be transferred to the KS active site and serve as the "starter" molecule for the initial elongation (condensation) reaction. All PUFA synthase systems identified to date contain a CLF domain and are part of a multidomain protein. Polypeptides (or domains of polypeptides) can be readily identified as belonging to the CLF family by sequence homology to known PUFA synthase CLFs.

A polypeptide or polypeptide domain having acyltransferase (AT) activity has been previously defined as having one or more of the following activities or characteristics: (1) the ability to transfer a fatty acyl group from an ACP domain to water (i.e., a thioesterase), releasing the fatty acyl group as a free fatty acid;

(2) can transfer fatty acyl groups to acceptors such as CoA,

(3) Acyl groups can be transferred between different ACP domains,

Alternatively (4) the fatty acyl group may be transferred to a lipophilic acceptor molecule (eg, to lysophosphatidic acid). Polypeptides (or domains of polypeptides) belonging to the AT family can be readily identified by sequence homology to known PUFA synthases, AT.

Polypeptides or polypeptide domains having enoyl-ACP reductase (ER) biological activity have previously been shown to reduce the trans double bond (introduced by DH activity) in fatty acyl-ACP, thereby saturating the associated carbon. ER domains in the PUFA synthase system have previously been shown to be homologous to the ER enzyme family (Heath et al., Nature 406:145-146 (2000), incorporated by reference in its entirety), and ER homologs have been shown to function as enoyl-ACP reductases in vitro (Bumpus et al., J. Am. Chem. Soc., 130:11614-11616 (2008), incorporated by reference in its entirety). The term "enoyl-ACP reductase" is used interchangeably with "enoyl reductase," "enoyl ACP-reductase," and "enoyl-ACP reductase." Polypeptides (or domains of polypeptides) belonging to the ER family can be readily identified by sequence homology to known PUFA synthases.

In some embodiments, the invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA1 (SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120). In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA1 (SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120), such as a KS domain (SEQ ID NO: 7 or SEQ ID NO: 74), a MAT domain (SEQ ID NO: 9 or SEQ ID NO: 76), an ACP domain (such as any of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACP domains, including a tandem domain (SEQ ID NO: 11 or SEQ ID NO: 78 and portions thereof), a KR domain (SEQ ID NO: 25 or SEQ ID NO: 100), a DH domain (SEQ ID NO: 13), or a combination of two or more ACP domains. NO: 27 or SEQ ID NO: 118) and combinations thereof. In some embodiments, the nucleic acid molecule comprises two or more polynucleotide sequences, wherein at least two or more polynucleotide sequences are each 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA1 (SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120). In some embodiments, the at least two or more polynucleotide sequences are 80% identical to the same polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to a different polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to different polynucleotide sequences in SEQ ID NO: 1, SEQ ID NO: 68 or SEQ ID NO: 120, and the order of the at least two or more polynucleotide sequences in the nucleic acid molecule is the same or different compared to the order of the corresponding sequences in SEQ ID NO: 1, SEQ ID NO: 68 or SEQ ID NO: 120. In some embodiments, the at least two or more polynucleotide sequences are each at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA1 (SEQ ID NO: 1, SEQ ID NO: 68 or SEQ ID NO: 120), such as a KS domain (SEQ ID NO: 7 or SEQ ID NO: 74), a MAT domain (SEQ ID NO: 9 or SEQ ID NO: 76), an ACP domain (such as any of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 80, 82, 84, 86, 88, 90, 92, 94, 96 or 98), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9 or 10 ACP domains, including a tandem domain (SEQ ID NO: 11 or SEQ ID NO: 78 and portions thereof), a KR domain (SEQ ID NO: 25 or SEQ ID NO: 26), or a tandem domain (SEQ ID NO: 12 or SEQ ID NO: 13). NO: 100), DH domain (SEQ ID NO: 27 or SEQ ID NO: 118), and combinations thereof. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding one or more PUFA synthase domains of PFA1 (SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA2 (SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121). In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA2 (SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121), such as a KS domain (SEQ ID NO: 29 or SEQ ID NO: 102), a CLF domain (SEQ ID NO: 31 or SEQ ID NO: 104), an AT domain (SEQ ID NO: 33 or SEQ ID NO: 106), an ER domain (SEQ ID NO: 35 or SEQ ID NO: 108), and combinations thereof. In some embodiments, the nucleic acid molecule comprises two or more polynucleotide sequences, wherein at least two or more polynucleotide sequences are 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA2 (SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121). In some embodiments, the at least two or more polynucleotide sequences are 80% identical to the same polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to a different polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to a different polynucleotide sequence in SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121, and the at least two or more polynucleotide sequences are arranged in the same or different order in the nucleic acid molecule compared to the order of the corresponding sequences in SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121. In some embodiments, the at least two or more polynucleotide sequences are each 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA2 (SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121), such as the KS domain (SEQ ID NO: 29 or SEQ ID NO: 102), the CLF domain (SEQ ID NO: 31 or SEQ ID NO: 104), the AT domain (SEQ ID NO: 33 or SEQ ID NO: 106), the ER domain (SEQ ID NO: 35 or SEQ ID NO: 108), and combinations thereof. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding one or more PUFA synthase domains of PFA2 (SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA3 (SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122). In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains of PFA3 (SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122), such as a DH domain (SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 110, or SEQ ID NO: 112), an ER domain (SEQ ID NO: 41 or SEQ ID NO: 114), and combinations thereof. In some embodiments, the nucleic acid molecule comprises two or more polynucleotide sequences, wherein at least two or more polynucleotide sequences are 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA3 (SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122). In some embodiments, the at least two or more polynucleotide sequences are 80% identical to the same polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to a different polynucleotide sequence encoding one or more PUFA synthase domains in SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122. In some embodiments, the at least two or more polynucleotide sequences are 80% identical to a different polynucleotide sequence in SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122, and the at least two or more polynucleotide sequences are arranged in the same or different order in the nucleic acid molecule compared to the order of the corresponding sequences in SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122. In some embodiments, the at least two or more polynucleotide sequences are each 80% identical to a polynucleotide sequence encoding one or more PUFA synthase domains in PFA3 (SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122), such as the DH domain (SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 110, or SEQ ID NO: 112), the ER domain (SEQ ID NO: 41 or SEQ ID NO: 114), and combinations thereof. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding one or more PUFA synthase domains of PFA3 (SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 68, or SEQ ID NO: 120, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 74, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 9 or SEQ ID NO: 76, wherein the polynucleotide sequence encodes a polypeptide comprising MAT activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to any one of SEQ ID NO: 13, 15, 17, 19, 21, 23, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity;

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 11 or SEQ ID NO: 78, wherein the polynucleotide sequence encodes a polypeptide comprising ACP activity.

In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one, two, three, four, five, or six ACP domains of SEQ ID NO: 11, wherein the polynucleotide sequence encodes a polypeptide comprising an ACP activity associated with one or more ACP domains. SEQ ID NOs: 13, 15, 17, 19, 21, and 23 are representative polynucleotide sequences encoding one ACP domain of SEQ ID NO: 11.

In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding one, two, three, four, five, six, seven, eight, nine, or ten ACP domains of SEQ ID NO: 78, wherein the polynucleotide sequence encodes a polypeptide comprising an ACP activity associated with one or more ACP domains. SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98 are representative polynucleotide sequences encoding one ACP domain of SEQ ID NO: 78.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 25 or SEQ ID NO: 100, wherein the polynucleotide sequence encodes a polypeptide comprising KR activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 27 or SEQ ID NO: 118, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 3, SEQ ID NO: 70, or SEQ ID NO: 121, wherein the polynucleotide sequence encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 29 or SEQ ID NO: 102, wherein the polynucleotide sequence encodes a polypeptide comprising KS activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 31 or SEQ ID NO: 104, wherein the polynucleotide sequence encodes a polypeptide comprising CLF activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 33 or SEQ ID NO: 106, wherein the polynucleotide sequence encodes a polypeptide comprising AT activity.

In some embodiments, the invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 35 or SEQ ID NO: 108, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to any one of SEQ ID NO: 5, SEQ ID NO: 72, or SEQ ID NO: 122, wherein the polynucleotide encodes a polypeptide comprising a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and a combination thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 37, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 39, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 110, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 112, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity.

In some embodiments, the invention relates to a nucleic acid molecule comprising a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 41 or SEQ ID NO: 114, wherein the polynucleotide sequence encodes a polypeptide comprising ER activity.

The present invention relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71), or Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), wherein the polynucleotide encodes a polypeptide comprising one or more PUFA synthase activities.

The present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of one or more PUFA synthase domains of a PUFA synthase of the present invention.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence within Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO: 8 or SEQ ID NO: 75), a MAT domain (SEQ ID NO: 10 or SEQ ID NO: 77), an ACP domain (such as any of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACP domains, including a tandem domain (SEQ ID NO: 12 or SEQ ID NO: 79 and portions thereof), a KR domain (SEQ ID NO: 26 or SEQ ID NO: 101), a DH domain (SEQ ID NO: 28 or SEQ ID NO: 29), or a DH domain (SEQ ID NO: 30). NO: 119) and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence comprising one or more PUFA synthase domains in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence comprising one or more PUFA synthase domains in Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69) that each comprise one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), and the at least two or more amino acid sequences are arranged in the same or different order in the polypeptide compared to the order of the corresponding domains in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to an amino acid sequence of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO: 8 or SEQ ID NO: 75), a MAT domain (SEQ ID NO: 10 or SEQ ID NO: 77), an ACP domain (such as any of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACP domains, including a tandem domain (SEQ ID NO: 12 or SEQ ID NO: 79 and portions thereof), a KR domain (SEQ ID NO: 26 or SEQ ID NO: 101), a DH domain (SEQ ID NO: 28 or SEQ ID NO: 29), or a DH domain (SEQ ID NO: 30). In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence within Pfa2p (SEQ ID NO:4 or SEQ ID NO:71) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence within Pfa2p (SEQ ID NO:4 or SEQ ID NO:71) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO:30 or SEQ ID NO:103), a CLF domain (SEQ ID NO:32 or SEQ ID NO:105), an AT domain (SEQ ID NO:34 or SEQ ID NO:107), an ER domain (SEQ ID NO:36 or SEQ ID NO:109), and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71) that comprises one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence in Pfa2p (SEQ ID NO: 4, SEQ ID NO: 71). In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa2p (SEQ ID NO: 4, SEQ ID NO: 71) that each comprise one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71), and the at least two or more amino acid sequences are arranged in the same or different order in the polypeptide compared to the order of the corresponding domains in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71). In some embodiments, the at least two or more amino acid sequences are 80% identical to an amino acid sequence comprising one or more PUFA synthase domains of Pfa2p (SEQ ID NO:4 or SEQ ID NO:71), such as a KS domain (SEQ ID NO:30 or SEQ ID NO:103), a CLF domain (SEQ ID NO:32 or SEQ ID NO:105), an AT domain (SEQ ID NO:34 or SEQ ID NO:107), an ER domain (SEQ ID NO:36 or SEQ ID NO:109), and combinations thereof. In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa2p (SEQ ID NO:4 or SEQ ID NO:71), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence within Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence within Pfa3 (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains, such as the DH domain (SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 111, or SEQ ID NO: 113), the ER domain (SEQ ID NO: 42 or SEQ ID NO: 115), and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence within Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence in Pfa3p (SEQ ID NO: 6, SEQ ID NO: 73) that comprises one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa3p (SEQ ID NO: 6, SEQ ID NO: 73) that each comprise one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), and the at least two or more amino acid sequences are arranged in the same or different order in the polypeptide compared to the order of the corresponding domains in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73). In some embodiments, the at least two or more amino acid sequences are 80% identical to an amino acid sequence comprising one or more PUFA synthase domains of Pfa3 (SEQ ID NO: 6 or SEQ ID NO: 73), such as the DH domain (SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 111, or SEQ ID NO: 113), the ER domain (SEQ ID NO: 42 or SEQ ID NO: 115), and combinations thereof. In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or SEQ ID NO: 69, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 8 or SEQ ID NO: 75, and the polypeptide comprises KS activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 10 or SEQ ID NO: 77, and the polypeptide comprises MAT activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14, 16, 18, 20, 22, 24, 81, 83, 87, 89, 91, 93, 95, 97 or 99, and the polypeptide comprises ACP activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 12 or SEQ ID NO: 79, and the polypeptide comprises ACP activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 12, and the polypeptide comprises ACP activity. In some embodiments, the amino acid sequence is at least 80% identical to an amino acid sequence comprising one, two, three, four, five, or six ACP domains in SEQ ID NO: 12, wherein the polypeptide comprises ACP activity associated with one or more ACP domains. SEQ ID NOs: 14, 16, 18, 20, 22, and 24 are representative amino acid sequences of SEQ ID NO: 12 comprising a single ACP domain.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 79, and the polypeptide comprises ACP activity. In some embodiments, the amino acid sequence is at least 80% identical to an amino acid sequence comprising one, two, three, four, five, six, seven, eight, nine, or ten ACP domains in SEQ ID NO: 79, wherein the polypeptide comprises ACP activity associated with one or more ACP domains. SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99 are representative amino acid sequences comprising individual ACP domains in SEQ ID NO: 79.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 26 or SEQ ID NO: 101, and the polypeptide comprises KR activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 28 or SEQ ID NO: 119, and the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 4 or SEQ ID NO: 71, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, CLF activity, AT activity, ER activity, and combinations thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 30 or SEQ ID NO: 103, and the polypeptide comprises KS activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 32 or SEQ ID NO: 105, and the polypeptide comprises CLF activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 34 or SEQ ID NO: 107, and the polypeptide comprises AT activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 36 or SEQ ID NO: 109, and the polypeptide comprises ER activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 6 or SEQ ID NO: 73, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and a combination thereof.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 38, and the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 40, and the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 111, and the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 113, and the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 42 or SEQ ID NO: 115, and the polypeptide comprises ER activity.

In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that is at least about 80%, 85%, or 90% identical to the polynucleotide sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequences reported herein. The term "percent identity" as known in the art refers to the relationship between two or more amino acid sequences or two or more nucleotide sequences as determined by sequence comparison. "Identical" in the art also refers to the degree of sequence relatedness between amino acid or polynucleotide sequences, as determined by pairing between word strings of such sequences, as appropriate.

For example, a polynucleotide sequence of a nucleic acid molecule is 95% "identical" to a reference polynucleotide sequence of the present invention, which means that the polynucleotide sequence of the nucleic acid molecule is identical to the reference sequence, except that the polynucleotide sequence differs from the reference polynucleotide sequence by up to 5 nucleotides per 100 nucleotides. In other words, to obtain a polynucleotide sequence that is at least 95% identical to the reference polynucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.

In practice, any particular polynucleotide sequence or amino acid sequence is typically determined by known computer programs to be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide sequence or amino acid sequence of the invention. Methods for determining the best overall match between a query sequence (a sequence of the invention) and a subject sequence can be performed by aligning the sequences and calculating an identity score. Alignments are performed using the computer program AlignX, a component of the Vector NTI suite from Invitrogen (www.invitrogen.com). Amino acid and polynucleotide sequences are aligned using ClustalW alignment software (Thompson, J.D., et al. Nucl. Acids Res. 22:4673-4680 (1994)). Amino acid and polynucleotide sequence alignments are performed using the default scoring matrices Blosum62mt2 and swgapdnamt, respectively. For amino acid sequences, the default gap opening penalty is 10 and the gap extension penalty is 0.1. For polynucleotide sequences, the default gap opening penalty is 15 and the gap extension penalty is 6.66.

The present invention relates to an isolated nucleic acid molecule comprising a polynucleotide encoding a polypeptide comprising PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, CLF activity, AT activity, ER activity, DH activity, and combinations thereof, wherein the polynucleotide hybridizes under stringent conditions to the complement of any of the above polynucleotide sequences.

A nucleic acid molecule is "hybridizable" with another nucleic acid molecule (e.g., cDNA, genomic DNA, or RNA) when the single-stranded form of the molecule can anneal to the other nucleic acid molecule under appropriate temperature and solution conditions. Hybridization and wash conditions are well known and readily exemplified. See, e.g., Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Temperature and ionic strength conditions determine the "stringency" of the hybridization. Stringent conditions can be adjusted to screen for moderately similar fragments, such as homologous fragments from distantly related organisms, to highly similar fragments, such as functionally duplicated enzymes from closely related organisms. Post-hybridization washes determine the stringency conditions. One set of conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 minutes, followed by repeated washes with 2X SSC, 0.5% SDS at 45°C for 30 minutes, and then two more washes with 0.2X SSC, 0.5% SDS at 50°C for 30 minutes. For more stringent conditions, washes are performed at higher temperatures, using the same conditions as above, except that the temperature for the final two 30-minute washes with 0.2X SSC, 0.5% SDS is increased to 60°C. Another set of highly stringent conditions has the final two washes at 0.1X SSC, 0.1% SDS at 65°C. Another highly stringent condition involves hybridization at 0.1X SSC, 0.1% SDS at 65°C, followed by washes with 2X SSC, 0.1% SDS, followed by 0.1X SSC, 0.1% SDS.

The present invention relates to isolated nucleic acid molecules containing polynucleotide sequences that are completely complementary to any of the polynucleotide sequences described above. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, when referring to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine.

In certain embodiments, the polynucleotide or nucleic acid is DNA. When referring to DNA, a nucleic acid molecule comprising a polypeptide-encoding polynucleotide sequence typically includes a promoter and/or other transcriptional or translational control elements operably linked to one or more coding regions. Operably linked means that when the coding region for a gene product, such as a polypeptide, is linked to one or more regulatory sequences in such a manner, the expression of the gene product is influenced or controlled by the regulatory sequences. Two DNA fragments (e.g., a polypeptide-encoding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or to transcribe the DNA template. Thus, a promoter region is operably linked to a polypeptide-encoding polynucleotide sequence if the promoter is capable of influencing transcription of the polynucleotide sequence. Promoters can be cell-specific promoters that direct substantial transcription of DNA only in predetermined cells. Typically, the coding region is located at the 3' terminus of the promoter. Promoters can be derived in their entirety from a natural gene, be composed of different elements from different natural promoters, or comprise synthetic DNA fragments. Those skilled in the art understand that different promoters can direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that often cause gene expression in most cell types are referred to as "constitutive promoters." It should also be recognized that, since regulatory sequence boundaries are often not completely defined, DNA fragments of different lengths can have the same promoter activity. The promoter of a gene is typically defined by the transcription start site at the 3' end, extending upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at levels detectable above background. Within the promoter, the transcription start site (often determined by nuclease S1 mapping) and the protein binding domain (consensus sequence) responsible for RNA polymerase binding can be found.

Suitable regulatory regions include nucleic acid regions located upstream (5' non-coding sequences), within the coding region, or downstream (3' non-coding sequences) that affect the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures. In addition to promoters, other transcriptional control elements include enhancers, operators, repressors, and transcription termination signals that can be operably linked to polynucleotides to direct cell-specific transcription. The boundaries of the coding sequence are defined by a 5' (amino) terminal translation start codon and a 3' (carboxyl) terminal translation stop codon. Coding regions include, but are not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is expressed in eukaryotic cells, a polyadenylation signal and a transcription termination sequence are often present 3' of the coding region.

In certain aspects of the present invention, polynucleotide sequences of at least 20 bases, at least 30 bases, or at least 50 bases that hybridize to the polynucleotide sequences of the present invention can be used as PCR primers. Typically, in PCR-type amplification techniques, primers have different sequences and are not complementary to each other. Depending on the desired detection conditions, the design of the primer sequence should ensure efficient and fidelity replication of the target nucleic acid. PCR primer design methods are common and well known in the art. Typically, when amplifying longer nucleic acid fragments encoding homologous genes from DNA or RNA, a polymerase chain reaction (PCR) protocol can be used to amplify two short fragments of the sequence. The polymerase chain reaction can also be performed on a library of cloned nucleic acid fragments, where the sequence of one primer is derived from the nucleic acid fragment and the sequence of the other primer is conveniently adapted to utilize the presence of a polyadenylic acid channel at the 3' end of the mRNA precursor encoding the microbial gene. Alternatively, the second primer sequence is based on a sequence from a cloning vector.

In addition, specific primers are designed and used to amplify a portion or the full length of the sequence. The resulting amplified product can be labeled directly during the amplification reaction, or it can be labeled after the amplification reaction and the separated full-length DNA fragments can be detected under conditions of appropriate stringency.

Thus, the nucleic acid molecules of the present invention can be used to isolate genes encoding homologous proteins from the same or other species or bacterial species. The use of sequence-dependent protocols for isolating homologous genes is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, nucleic acid hybridization methods, as exemplified by the various uses of nucleic acid amplification technology, and DNA and RNA amplification methods (e.g., polymerase chain reaction, Mullis et al., U.S. Patent No. 4,683,202; ligase chain reaction (LCR) (Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985)); or strand displacement amplification (SDA; Walker et al., Proc. Natl. Acad. Sci. U.S.A. 89:392 (1992)).

In some embodiments, the isolated nucleic acid molecules of the present invention are used to isolate homologous nucleic acid molecules from other organisms to identify PUFA synthases that produce similar or improved PUFA profiles. In some embodiments, the isolated nucleic acid molecules of the present invention are used to isolate homologous nucleic acid molecules from other organisms that are involved in producing large amounts of DHA.

The nucleic acid molecules of the present invention further comprise a polynucleotide sequence encoding a PUFA synthase gene, a PUFA synthase gene domain, or a PUFA synthase gene fragment fused in frame to a marker sequence for detecting the polypeptides of the present invention. Marker sequences include auxotrophic or dominant markers known to those of ordinary skill in the art, such as ZEO (zeromycin), NEO (G418), hygromycin, arsenite, HPH, NAT, and the like.

The present invention also encompasses variants of PUFA synthase genes. Variants may include alterations in the coding region, noncoding region, or both. Examples of polynucleotide variants include changes that produce silent substitutions, additions, or deletions that do not alter the properties or activity of the encoded polypeptide. In certain embodiments, polynucleotide sequence variants are produced by silent substitutions due to degeneracy in the gene codon. In some embodiments, polynucleotide sequences are generated for a variety of reasons, such as, for example, optimizing codons for expression in a specific host (e.g., changing codons in Thraustochytrid to codons preferred in other organisms, such as E. coli or Saccharomyces cerevisiae).

The present invention also provides allelic variants, orthologs and/or species homologs. Using the sequence information disclosed herein, procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs and/or species homologs of the genes described herein. For example, allelic variants and/or species homologs can be isolated and identified by generating suitable probes or primers based on the sequences provided herein and screening for suitable nucleic acid sources for allelic variants and/or desired homologs.

The present invention relates to a recombinant nucleic acid molecule comprising any of the above nucleic acid molecules or a combination thereof and a transcription control sequence. In some embodiments, the recombinant nucleic acid molecule is a recombinant vector.

The present invention relates to a method for producing a recombinant vector comprising inserting one or more of the isolated nucleic acid molecules described herein into a vector.

The vector of the present invention can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, or the like.

The polynucleotide sequences of the present invention may be contained in any expression vector for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal, and synthetic DNA or RNA sequences, such as SV40 derivatives; bacterial plasmids; and yeast plasmids. However, any other suitable vector known to those of ordinary skill in the art may be used.

Can by various steps, suitable dna sequence is inserted into carrier.Usually, dna sequence is inserted into suitable restriction endonuclease cleavage site by methods known in the art.Such steps and other can be considered as within the scope known to those skilled in the art.

The present invention also includes recombinant constructs containing one or more of the above-mentioned polynucleotide sequences. The constructs include vectors, such as plasmids or viral vectors, into which one or more sequences of the present invention are inserted in either a forward or reverse orientation. In one aspect of the invention, the constructs also include regulatory sequences, including, for example, promoters operably linked to the sequences. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available.

peptides

The present invention relates to isolated polypeptides comprising the amino acid sequence of a PUFA synthase protein and domain from the isolated microorganisms with ATCC Accession Nos. PTA-9695 and PTA-10212.

As used herein, the term "polypeptide" includes both singular and plural forms, encompassing molecules composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, rather than a product of a specific length. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be substituted or used interchangeably with these terms. The term "polypeptide" also refers to products of modifications after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amination, derivatization by known protecting/blocking groups, protease cleavage, or modification with non-natural amino acids.

The polypeptides described herein include, but are not limited to, fragments, variants, or derivative molecules. The terms "fragment," "variant," "derivative," and "analog," when referring to polypeptides, include any polypeptide that retains at least some biological activity. Polypeptide fragments may include any proteolytic fragments, deletion fragments, and fragments that readily reach the site of action when delivered to an animal. Polypeptide fragments also include any portion of a polypeptide that comprises an antigenic or immunogenic epitope of a native polypeptide, including linear or three-dimensional epitopes. Polypeptide fragments may comprise variant regions, including those described above, as well as polypeptides whose amino acid sequence is altered by amino acid substitutions, deletions, or insertions. Variants may occur naturally, such as allelic variants. By "allelic variant" is meant an alternative form of a gene occupying a given locus on an organism's chromosome. Non-natural variants are generated using mutagenesis techniques known in the art. Polypeptide fragments of the present invention may comprise conservative or non-conservative amino acid substitutions, deletions, or additions. Polypeptide variants are also referred to herein as "polypeptide analogs." Polypeptide fragments of the present invention include derivative molecules. As used herein, a "derivative" of a polypeptide or polypeptide fragment refers to one or more residues of the subject polypeptide chemically derivatized by reacting functional side chains. "Derivatives" also include peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

The polypeptides of the present invention may be encoded by any nucleic acid molecule of the present invention.

The present invention relates to an isolated polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71), Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), and combinations thereof, wherein the polypeptide comprises one or more PUFA synthase activities.

The present invention relates to polypeptides comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of one or more PUFA synthase domains of a PUFA synthase of the present invention.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence of Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO: 8 or SEQ ID NO: 75), a MAT domain (SEQ ID NO: 10 or SEQ ID NO: 77), an ACP domain (such as any of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACP domains, including a tandem domain (SEQ ID NO: 12 or SEQ ID NO: 79 and portions thereof), a KR domain (SEQ ID NO: 26 or SEQ ID NO: 101), a DH domain (SEQ ID NO: 28 or SEQ ID NO: 29), or a DH domain (SEQ ID NO: 30). NO: 119) and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence comprising one or more PUFA synthase domains in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence comprising one or more PUFA synthase domains in Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to a different amino acid sequence comprising one or more PUFA synthase domains in Pfa1p (SEQ ID NO: 2, SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are 80% identical to a different amino acid sequence in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), and the at least two or more amino acid sequences are arranged in the same or a different order in the polypeptide compared to the order of the corresponding domains in Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69). In some embodiments, the at least two or more amino acid sequences are at least 80% identical to an amino acid sequence of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO: 8 or SEQ ID NO: 75), a MAT domain (SEQ ID NO: 10 or SEQ ID NO: 77), an ACP domain (such as any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 81, 83, 85, 87, 89, 91, 93, 95, 97, or 99), a combination of two or more ACP domains, such as a combination of 2, 3, 4, 5, 6, 7, 8, 9, or 10 ACP domains, including a tandem domain (SEQ ID NO: 12 or SEQ ID NO: 79 and portions thereof), a KR domain (SEQ ID NO: 26 or SEQ ID NO: 101), a DH domain (SEQ ID NO: 28 or SEQ ID NO: 29), or a DH domain (SEQ ID NO: 30). In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa1p (SEQ ID NO: 2 or SEQ ID NO: 69), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71) comprising one or more PUFA synthase domains, such as a KS domain (SEQ ID NO: 30 or SEQ ID NO: 103), a CLF domain (SEQ ID NO: 32 or SEQ ID NO: 105), an AT domain (SEQ ID NO: 34 or SEQ ID NO: 107), an ER domain (SEQ ID NO: 36 or SEQ ID NO: 109), and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71) comprising one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence in Pfa2p (SEQ ID NO: 4, SEQ ID NO: 71). In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa2p (SEQ ID NO: 4, SEQ ID NO: 71), each comprising one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71), and the at least two or more amino acid sequences are arranged in the same or different order in the polypeptide compared to the order of the corresponding domains in Pfa2p (SEQ ID NO: 4 or SEQ ID NO: 71). In some embodiments, the at least two or more polypeptide molecules are 80% identical to an amino acid sequence comprising one or more PUFA synthase domains of Pfa2p (SEQ ID NO:4 or SEQ ID NO:71), such as the KS domain (SEQ ID NO:30 or SEQ ID NO:103), the CLF domain (SEQ ID NO:32 or SEQ ID NO:105), the AT domain (SEQ ID NO:24 or SEQ ID NO:107), the ER domain (SEQ ID NO:36 or SEQ ID NO:109), and combinations thereof. In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa2p (SEQ ID NO:4 or SEQ ID NO:71), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80% identical to an amino acid sequence in Pfa3 (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains, such as a DH domain (SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 111, or SEQ ID NO: 113), an ER domain (SEQ ID NO: 42 or SEQ ID NO: 115), and combinations thereof. In some embodiments, the polypeptide comprises two or more amino acid sequences, wherein each of the at least two or more amino acid sequences is 80% identical to an amino acid sequence in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73) comprising one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to the same amino acid sequence in Pfa3p (SEQ ID NO: 6, SEQ ID NO: 73) that comprises one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa3p (SEQ ID NO: 6, SEQ ID NO: 73) that each comprise one or more PUFA synthase domains. In some embodiments, the at least two or more amino acid sequences are 80% identical to different amino acid sequences in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), and the at least two or more amino acid sequences are arranged in the same or different order in the polypeptide compared to the order of the corresponding domains in Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73). In some embodiments, the at least two or more amino acid sequences are 80% identical to an amino acid sequence comprising one or more PUFA synthase domains of Pfa3 (SEQ ID NO: 6 or SEQ ID NO: 73), such as the DH domain (SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 111, or SEQ ID NO: 113), the ER domain (SEQ ID NO: 42 or SEQ ID NO: 115), and combinations thereof. In some embodiments, the polypeptide comprises one or more amino acid sequences comprising one or more PUFA synthase domains of Pfa3p (SEQ ID NO: 6 or SEQ ID NO: 73), including one or more copies of any individual domain in combination with one or more copies of any other individual domain.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2 or SEQ ID NO: 69, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of KS activity, MAT activity, ACP activity, KR activity, DH activity, and combinations thereof.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 8 or SEQ ID NO: 75, wherein the polypeptide comprises KS activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or SEQ ID NO: 77, wherein the polypeptide comprises MAT activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 14, 16, 18, 20, 22, 24, 81, 83, 87, 89, 91, 93, 95, 97, or 99, and the polypeptide comprises ACP activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 12 or SEQ ID NO: 79, wherein the polypeptide comprises ACP activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 12, wherein the polypeptide comprises ACP activity. In some embodiments, the amino acid sequence is at least 80% identical to an amino acid sequence comprising one, two, three, four, five, or six ACP domains in SEQ ID NO: 12, wherein the polypeptide comprises ACP activity associated with one or more ACP domains. SEQ ID NOs: 14, 16, 18, 20, 22, and 24 are representative amino acid sequences of SEQ ID NO: 12 comprising a single ACP domain.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 79, wherein the polypeptide comprises ACP activity. In some embodiments, the amino acid sequence is at least 80% identical to an amino acid sequence comprising one, two, three, four, five, six, seven, eight, nine, or ten ACP domains in SEQ ID NO: 79, wherein the polypeptide comprises ACP activity associated with one or more ACP domains. SEQ ID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97, and 99 are representative amino acid sequences comprising individual ACP domains in SEQ ID NO: 79.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 26 or SEQ ID NO: 101, wherein the polypeptide comprises KR activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 28 or SEQ ID NO: 119, wherein the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 4 or SEQ ID NO: 71, and the polypeptide comprises a PUFA synthase activity selected from KS activity, CLF activity, AT activity, ER activity, and combinations thereof.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 30 or SEQ ID NO: 103, wherein the polypeptide comprises KS activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 32 or SEQ ID NO: 105, wherein the polypeptide comprises CLF activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 34 or SEQ ID NO: 107, wherein the polypeptide comprises AT activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 36 or SEQ ID NO: 109, wherein the polypeptide comprises ER activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 6 or SEQ ID NO: 73, and the polypeptide comprises a PUFA synthase activity selected from the group consisting of DH activity, ER activity, and combinations thereof.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 38, wherein the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 40, wherein the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 111, wherein the polypeptide comprises DH activity.

In some embodiments, the present invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 113, wherein the polypeptide comprises DH activity.

In some embodiments, the invention relates to a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 42 or SEQ ID NO: 115, wherein the polypeptide comprises ER activity.

In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 80%, 85%, or 90% identical, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences reported herein.

The amino acid sequence of a polypeptide is at least, for example, 95% "identical" to a query amino acid sequence, which means that the amino acid sequence of the subject polypeptide is identical to the query sequence, except that the subject polypeptide sequence may include up to 5 amino acid changes for every 100 amino acids in the query amino acid sequence. In other words, to obtain a polypeptide comprising an amino acid sequence at least 95% identical to the query amino acid sequence, up to 5% of the amino acids in the subject sequence may be inserted, deleted, (gained or lost positions), or substituted with other amino acids. These changes in the reference sequence can occur at the amino or carboxyl termini of the reference amino acid sequence or anywhere between these termini, and can be scattered throughout the reference sequence or occur in one or more contiguous sequences within the reference sequence.

In practice, whether any particular polypeptide has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of the invention is generally determined by known computer programs. As described above, methods for determining the best overall match between a query sequence (a sequence of the invention) and a subject sequence can be performed by aligning the sequences and calculating an identity score. Alignments are performed using the computer program AlignX, which is a component of the Vector NTI suite from Invitrogen (www.invitrogen.com). Alignments are performed using ClustalW alignment software (J. Thompson et al., Nucleic Acids Res. 22(22): 4673-4680 (1994)).

The default scoring matrix Blosum62mt2 is used. The default gap opening penalty is 10 and the default gap extension penalty is 0.1.

In other aspects of the invention, nucleic acid molecules having a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide sequence disclosed herein encode a polypeptide having one or more PUFA synthase activities. Polypeptides having one or more PUFA synthase activities exhibit one or more activities that are similar, but not necessarily identical, to one or more activities of a PUFA synthase of the invention.

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will readily recognize that most nucleic acid molecules having a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences described herein encode polypeptides that "have PUFA synthase functional activity." In fact, since degenerate variants of any of these polynucleotides encode the same polypeptide, in many cases, one skilled in the art can predict a polypeptide that exhibits activity based on conservative substitutions and knowledge of conserved functional domains. In certain aspects of the present invention, the polypeptides and polynucleotides of the present invention are provided in isolated form, e.g., purified to homogeneity. Alternatively, the polypeptides and polynucleotides of the present invention can be produced synthetically by conventional synthesizers.

It is known in the art that the "similarity" between two polypeptides can be determined by comparing the amino acid sequence and conservative amino acid substitutions of the polypeptide to the sequence of a second polypeptide.

In some embodiments, the polypeptides of the present invention are fusion polypeptides.

As used herein, "fusion polypeptide" refers to a polypeptide comprising a first polypeptide linearly connected to a second polypeptide by a peptide bond. The first polypeptide and the second polypeptide may be the same or different and may be directly connected or connected by a peptide linker. The terms "connected", "fused" and "fusion" as used herein may be used interchangeably. These terms refer to the binding of two or more elements or components together by some means, such as chemical coupling or recombination. "In-frame fusion" refers to the connection of two or more open reading frames to form a continuous longer open reading frame in a manner that maintains the correct reading frame of the original open reading frame. Thus, the resulting recombinant fusion protein is a single protein comprising two or more fragments corresponding to the polypeptides encoded by the original open reading frames (these fragments are not usually connected in this way under natural conditions). Although the reading frames are continuous fusion fragments, the fragments can still be physically or spatially divided, such as in-frame linker sequences. The "linker" sequence is one or more amino acids that separate the two polypeptide coding regions in the fusion protein.

The present invention relates to compositions comprising one or more polypeptides of the present invention and a biologically acceptable carrier.

In some embodiments, the composition comprises a biologically acceptable "excipient," wherein the excipient is a component or mixture of components that is used in the composition of the present invention to impart the desired characteristics to the composition, including a carrier. "Biologically acceptable" refers to a compound, material, composition, salt, and/or dosage form that, within the scope of adequate medical evaluation, is suitable for contact with living tissue without causing excessive toxicity, irritation, inflammatory response, or other problematic complications, and has a reasonable benefit/risk ratio during the contact period. Various excipients can be used. In some embodiments, an excipient can be, but is not limited to, an alkali agent, a stabilizer, an antioxidant, an adhesive, a separating agent, a finishing agent, an external phase component, a controlled release component, a solvent, a surfactant, a humectant, a buffer, a filler, a softener, and combinations thereof. In addition to the excipients described herein, excipients may also include, but are not limited to, those listed in Remington: The Science and Practice of Pharmacy, ^21st ed. (2005). The inclusion of an excipient in a specific category (e.g., "solvent") herein is intended to illustrate the role of the excipient, not to limit it. A particular excipient may fall into more than one classification.

The present invention also relates to fragments, variants, derivatives or analogs of any of the polypeptides disclosed herein.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide.

host cells

The present invention relates to host cells expressing any of the aforementioned nucleic acid molecules, any of the aforementioned recombinant nucleic acid molecules, and combinations thereof.

As used herein, the term "expression" refers to the process by which a gene produces a biochemical, such as RNA or a polypeptide. This process encompasses any functional manifestation of a gene within a cell, including, but not limited to, gene knockout, transient expression, and stable expression. This includes, but is not limited to, the transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of such mRNA into a polypeptide. If the desired end product is a biochemical, expression includes the creation of that biochemical and any precursors.

To produce one or more desired polyunsaturated fatty acids, host cells can be genetically modified to introduce the PUFA synthase system of the present invention into the host cells.

When an organism is genetically modified according to the present invention to express a PUFA synthase system, some host organisms may endogenously express accessory proteins required for the PUFA synthase system to produce PUFAs. However, even when an organism endogenously produces homologous accessory proteins, it may be necessary to transform the organism with a nucleic acid molecule encoding one or more accessory proteins to enable or enhance PUFA production. Some heterologous accessory proteins may work more effectively or efficiently with the transformed PUFA synthase protein than the host cell's endogenous accessory proteins.

Accessory proteins are defined herein as proteins that are not part of the core PUFA synthase system (i.e., not part of the PUFA synthase complex itself), but that may be necessary for the production or efficient production of PUFAs using the core PUFA synthase complex of the present invention. For example, to produce PUFAs, the PUFA synthase system must work with accessory proteins that transfer the 4'-phosphopantetheinyl moiety from coenzyme A to the acyl carrier protein (ACP) domain. Thus, a PUFA synthase system can be considered to include at least one 4'-phosphopantetheinyl transferase (PPTase) domain, or such a domain can be considered an accessory domain or protein of the PUFA synthase system. The structural and functional characteristics of PPTases are described in U.S. Patent Application Publication Nos. 2002/0194641, 2004/0235127, and 2005/0100995.

A domain or protein having the biological activity (function) of a 4'-phosphopantetheinyltransferase is characterized as an enzyme that transfers a 4'-phosphopantetheinyl moiety from coenzyme A to an acyl carrier protein (ACP). This transfer to an invariant serine residue in the ACP activates the inactive apo-form to the active holo-form. In the synthesis of polyketides and fatty acids, the phosphopantetheinyl group forms a thioester with the growing acyl chain. PPTases are a family of well-characterized enzymes involved in the synthesis of fatty acids, polyketides, and nonribosomal peptides. The sequences of several PPTases are known, crystal structures have been determined (e.g., Reuter K., et al., EMBO J. 18(23):6823-31 (1999)), and mutational analysis has identified several amino acid residues that are critical for activity (Mofid M.R. et al., Biochemistry 43(14):4128-36 (2004)).

A previous heterologous PPTase that recognized the Schizochytrium ACP domain as a substrate was the Het I protein from Nostoc sp. PCC7120 (formerly known as Anabaena sp. PCC 7120). Het I is a gene found in Nostoc sp. that is known to be responsible for the synthesis of long-chain hydroxy fatty acids, components of the glycolipid layer in the heterocysts of this organism (Black and Wolk, J. Bacteriol. 176:2282-2292 (1994); Campbell et al., Arch. Microbiol. 167:251-258 (1997)). Het I likely activates the ACP domain of Hgl E, a protein found in this class. Sequences and constructs containing Het I are described, for example, in U.S. Patent Application Publication No. 2007/0244192, which is incorporated by reference in its entirety.

Another heterologous PPTase previously demonstrated to recognize the Schizochytrium ACP domain is Sfp, from Bacillus subtilis. Sfp is well-studied and widely used because it recognizes a wide range of substrates. Based on published sequence information (Nakana et al., Molecular and General Genetics 232:313-321 (1992)), an expression vector for Sfp was previously generated by cloning the coding region and defined upstream and downstream flanking DNA sequences into the pACYC-184 cloning vector. This construct encodes a functional PPTase and has been shown to result in DHA accumulation in E. coli when co-expressed with Schizochytrium Orfs under appropriate conditions (see U.S. Application Publication No. 2004/0235127, incorporated by reference in its entirety).

Host cells include microbial cells, animal cells, plant cells, and insect cells. Representative examples of suitable hosts include bacterial cells; thermophilic or mesophilic bacteria; marine bacteria; thraustochytrids; fungal cells such as yeast; plant cells; insect cells; and isolated animal cells. Host cells can be untransfected or transfected with at least one other recombinant nucleic acid molecule. Host cells can also include transgenic cells that have been engineered to express a PUFA synthase. Those skilled in the art will be able to select a suitable host upon studying this disclosure.

Host cells include any microorganism of the order Thraustochytrids, such as microorganisms of genera including, but not limited to, Thraustochytrium, Labyrinthuloides, Japonochytrium, and Schizochytrium. Species within these genera include, but are not limited to, any species of Schizochytrium, including Schizochytrium aggregatum, Schizochytrium limacinum, and Schizochytrium minutum; any species of Thraustochytrium (including former species of the genus Ulkenia, such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiate, U. minuta, and Ulkenia sp.) BP-5601), including Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum; and species of Japonochytrium. Thraustochytrid strains include, but are not limited to, Schizochytrium (S31) (ATCC 20888), Schizochytrium (S8) (ATCC 20889), Schizochytrium (LC-RM) (ATCC 18915), Schizochytrium (SR21), Goldstein et Belsky (ATCC 28209), Honda et Yokochi (IFO 32693), Thraustochytrium (23B) (ATCC 20891), Stout (Schneider) (ATCC 24473), Goldstein (ATCC 34304), Rossa (Goldstein) (ATCC 28210), and Japonicum (L1) (ATCC 28207). Examples of other suitable genetically modified host microorganisms include, but are not limited to, yeast, including Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeasts, such as Candida, Kluyveromyces lactis, or other fungi, such as filamentous fungi such as Aspergillus, Neurospora, Penicillium, etc. Bacterial cells can also be used as hosts. Including Escherichia coli for fermentation processes. Alternatively, species such as Lactobacillus or Bacillus can be used as hosts.

Plant host cells include, but are not limited to, any higher plant, including dicots and monocots, as well as consumer plants, including crops and oilseed plants. These plants may include, for example, canola, soybean, rapeseed, linseed, corn, safflower, sunflower, and tobacco. Other preferred plants include plants known to produce compounds useful as pharmaceuticals, fragrances, nutrients, functional food ingredients, or cosmetic actives, or plants genetically engineered to produce such compounds/agents. Thus, any plant species or plant cell may be selected. Plants and plant cells, and plants from which they are derived, include, but are not limited to, plants and plant cells from Brassica rapa L.; Brassica cultivars NQC02CNX12 (ATCC PTA-6011), NQC02CNX21 (ATCC PTA-6644), and NQC02CNX25 (ATCC PTA-6012), and cultivars, cultivars, and plant parts derived from Brassica cultivars NQC02CNX12, NQC02CNX21, and NQC02CNX25 (see U.S. Patents 7,355,100, 7,456,340, and 7,348,473, respectively); soybean (Glycine max), rapeseed (Brassica spp.); linseed/flax (Linum usitatissimum), corn (Zea mays), safflower (Carthamus tinctorius); sunflower (Helianthus annuus); tobacco (Nicotiana tabacum); Arabidopsis thaliana, Brazil nut (Betholettia excelsa); castor bean (Riccinus communis), coconut (Cocus nucifera), coriander (Coriandrum sativum), cotton (Gossypium spp.), peanut (Arachis hypogaea); jojoba (Simmondsia chinensis); mustard tuber (Brassica spp. and Sinapis alba); oil palm (Elaeis guineeis), olive oil (Olea eurpaea); rice (Oryza sativa), pumpkin (Cucurbita maxima), barley (Hordeum vulgare), wheat (Traeticum aestivum); and duckweed (Lemnaceae sp.). Plant lines from these or other plants can be produced, selected or optimized to have desired traits, such as or related to, but not limited to, seed yield, lodging resistance, emergence, disease resistance or tolerance, maturity, late season plant integrity, plant height, resistance to defoliation, ease of plant modification, oil content or oil characteristics. Plant lines can be selected through plant breeding, such as pedigree breeding, recurrent selection breeding, intercross and backcross breeding, and such as marker-assisted breeding and farming methods. See, for example, U.S. Patent No. 7,348,473.

Animal cells include any isolated animal cell.

The present invention relates to host cells that express one or more nucleic acid molecules or recombinant nucleic acid molecules, including vectors, of the present invention.

The present invention relates to a method for preparing a recombinant host cell, comprising introducing a recombinant vector into the host cell.

Using the vectors of the present invention, such as cloning vectors or expression vectors, host cells can be genetically engineered (transduced, transformed, or transfected). The vectors can be in the form of, for example, plasmids, viral particles, bacteriophages, etc. The vectors contain the polynucleotide sequences described herein, as well as suitable promoters or control sequences, and can be used to transform suitable hosts to express the polypeptides encoded by the polynucleotide sequences. Genetic modification of host cells can also include genetic optimization for preferred or optimal host codon usage.

The engineered host cells can be cultured in conventional nutrient media modified for promoter activation, transformant selection, or amplification of the genes of the invention. Culture conditions, such as temperature, pH, and the like, and conditions for selecting host cells for expression, are known to those of ordinary skill in the art.

In some embodiments, the present invention relates to genetically modified plants or plant parts to express the PUFA synthase system described herein, which includes at least the core PUFA synthase enzyme complex. "Part of a plant" or "plant part," as defined herein, includes any part of a plant, such as, but not limited to, seeds (immature or mature), oil, pollen, embryos, flowers, fruits, buds, leaves, roots, stems, explants, and the like. In some embodiments, the genetically modified plant or plant part produces one or more PUFAs, such as EPA, DHA, DPA (n-3 or n-6), ARA, GLA, SDA, other PUFAs, and combinations thereof. Plants are not known to contain endogenous PUFA synthase systems; therefore, the PUFA synthase systems of the present invention can be used to engineer plants with unique fatty acid production capabilities. In other embodiments, the plant or plant part is further genetically modified to express at least one PUFA synthase accessory protein (e.g., a PPTase). In some embodiments, the plant is an oilseed plant, wherein the oilseed and/or the oil from the oilseed contains PUFAs produced by the PUFA synthase system. In some embodiments, the oil in the genetically modified plant, plant part, oilseed, and/or oilseed comprises a detectable amount of at least one PUFA produced by the PUFA synthase system. In other embodiments, the oil in such plant, plant part, oilseed, and/or oilseed is substantially free of intermediates or byproducts that are not the primary PUFA product of the introduced PUFA synthase system and that are not naturally produced by the endogenous FAS system of the wild-type plant. Although wild-type plants produce many short- and medium-chain PUFAs, such as 18-carbon PUFAs, through the FAS system, new or additional PUFAs can be produced in the oil of the plant, plant part, oilseed, and/or oilseed as a result of genetic modification using the PUFA synthase system described herein.

Plants can be genetically modified using classical plant development and/or molecular genetic techniques. See U.S. Application Publication No. 2007/0244192. Methods for producing transgenic plants are known in the art, wherein a recombinant nucleic acid molecule encoding a desired amino acid sequence is inserted into the plant's genome. For example, transgenic plants can be produced using viral vectors, such as by transforming monocots using viral vectors as described in U.S. Patent Nos. 5,569,597; 5,589,367; and 5,316,931. Methods for genetically engineering or modifying plants by transformation are well known in the art, including both biological and physical transformation protocols. See, for example, B.L. Miki et al., "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 67-88 (Glick, B.R. and Thompson, J.E., eds., CRC Publishing Co., Boca Raton, 1993). In addition, vectors and in vitro culture methods for transformation of plant cells or tissues and plant regeneration are available. See, for example, M.Y. Gruber et al., "Vectors for Plant Transformation," in METHODS IN PLANTMOLECULAR BIOLOGY AND BIOTECHNOLOGY 67-88 (Glick, B.R. and Thompson, J.E., eds., CRC Publishing Co., Boca Raton, 1993).

A widely used method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985) and U.S. Patent No. 6,051,757. Agrobacterium tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that can genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry the genes responsible for genetic transformation of plants. See, for example, Kado, C.I., Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods of Agrobacterium-mediated gene transfer are provided in several references, including Gruber et al., supra; Miki et al., supra; Moloney et al., Plant Cell Reports 2001; and others. 8:238 (1989); U.S. Patent Nos. 5,177,010; 5,104,310; 5,149,645; 5,469,976; 5,464,763; 4,940,838; 4,693,976; 5,591,616; 5,231,019; 5,463,174; 4,762,785; 5,004,863; and 5,159,135; and European Patent Application Nos. 0131624, 120516, 159418, 176112, 116718, 290799, 320500, 604662, 627752, 0267159, and 0292435.

Other methods for plant transformation include microprojectile-mediated transformation, in which the DNA is carried on the surface of the microprojectile. Biolistic devices are used to accelerate the microparticles to a velocity sufficient to penetrate plant cell walls and membranes in order to introduce the expression vector into plant tissue. See, Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, JC, Trends Biotech. 6:299 (1988), Sanford, JC, Physiol. Plant 79:206 (1990), Klein et al., Biotechnology 10:268 (1992), and U.S. Patents 5,015,580 and 5,322,783. Techniques for accelerating genetic material-coated microprojectiles into cells have also been described, such as those described in U.S. Patents 4,945,050 and 5,141,141. Another method for delivering DNA to plants by physical means is sonication of target cells. See, for example, Zhang et al., Bio/Technology 9:996 (1991). Alternatively, expression vectors can be introduced into plants using liposomes or spheroplast fusion. See, for example, Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNA into protoplasts using _CaCl precipitation, DNA injection, polyvinyl alcohol, or poly-L-ornithine has been reported. See, for example, Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and intact cells and tissues has also been reported. See, for example, Donn et al., Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24: 51-61 (1994); International Application Publication Nos. WO 87/06614, WO 92/09696 and WO 93/21335; and U.S. Pat. Nos. 5,472,869 and 5,384,253. Other transformation techniques include the whisker technique, see, for example, U.S. Pat. Nos. 5,302,523 and 5,464,765.

Chloroplasts or plastids can also be transformed directly. Thus, recombinant plants can be produced in which only chloroplast and plastid DNA is modified with any of the nucleic acid molecules and recombinant nucleic acid molecules described above, and combinations thereof. Promoters that function in chloroplasts or plastids are known in the art. See, for example, Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70 (1987). Methods and compositions for obtaining cells containing chloroplasts into which heterologous DNA has been inserted can be found, for example, in U.S. Patent Nos. 5,693,507 and 5,451,513.

Other methods for efficient transformation may also be used.

Suitable vectors for plant transformation are well known in the art. See, for example, U.S. Patent Nos. 6,495,738; 7,271,315; 7,348,473; 7,355,100; 7,456,340; 5,571,698 and 5,625,033, and references therein.

The expression vector may contain at least one genetic marker operably linked to a regulatory element (e.g., a promoter) so that transformed cells containing the marker can be recovered by negative selection (i.e., inhibiting the growth of cells lacking the marker gene) or positive selection (screening for the product encoded by the genetic marker). Several commonly used selectable marker genes are well known in the transformation art, such as genes encoding enzymes that metabolize and detoxify chemical agents such as antibiotics or herbicides, or genes that alter the target site, rendering it insensitive to the inhibitor. Selectable markers useful for plant transformation include, but are not limited to, the aminoglycoside phosphotransferase gene from the transposon Tn5 (AphII), which encodes resistance to kanamycin, neomycin, and G418, as well as genes encoding resistance or tolerance to glyphosate, hygromycin, methotrexate, bialophos, imidazolines, and sulfonylurea triazolopyrimidine sulfonamide herbicides such as chlorsulfuron, bromobenzene, and darabon. One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, which, under the control of plant regulatory signals, confers kanamycin resistance. See, for example, Fraley et al., Proc. Natl. Acad. Sci. U.S.A. 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin. See, for example, Vanden Elzen et al., Plant Mol. Biol. 5:299 (1985). Other resistance-conferring selectable marker genes of bacterial origin include gentamicin acetyltransferase, streptomycin phosphotransferase, aminoglycoside 3′-adenylate transferase, and the bleomycin resistance determinant. See Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet. 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil. See, for example, Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), and Stalker et al., Science 242:419-423 (1988). Other selectable marker genes for plant transformation are of non-bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactate synthase. See, for example, Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990).

Reporter genes can be used with or without a selectable marker. A reporter gene is a gene not normally present in the recipient organism that often encodes a protein that results in a phenotypic change or enzymatic characteristic. See, for example, K. Weising et al., Ann. Rev. Genetics 22:421 (1988). Examples of reporter genes include, but are not limited to, β-glucuronidase (GUS), β-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, and luciferase genes. See, for example, Jefferson, R.A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3:1681 (1984), and Chalfie et al., Science 263:802 (1994). An assay can be used to detect reporter gene expression at an appropriate time after the gene has been introduced into the recipient cells. Such an assay requires the use of the gene encoding the β-glucuronidase (GUS) gene from the β-glucuronidase locus of Escherichia coli, as described in Jefferson et al., Biochem. Soc. Trans. 15: 17-19 (1987).

Promoter regulatory elements from a variety of sources can be effectively used in plant cells to express exogenous genes. For example, promoter regulatory elements from bacterial sources, such as the octopine synthase promoter, nopaline synthase promoter, and mannopine synthase promoter, as well as promoters from viral sources, such as cauliflower mosaic virus (35S and 19S), 35T (this is a re-engineered 35S promoter, see International Application Publication No. WO97/13402). Plant promoter regulatory elements include, but are not limited to, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), β-conglycinin promoter, β-phaseolin promoter, ADH promoter, heat shock promoters, and tissue-specific promoters. Matrix linker regions, scaffold linker regions, introns, enhancers, and polyadenylation sequences can also be used to improve transcription efficiency and DNA integration. Such elements can be included to obtain the desired properties of transforming DNA in plants. Typical elements include, but are not limited to, Adh-intron 1, Adh-intron 6, alfalfa mosaic virus coat protein leader sequence, corn streak virus coat protein leader sequence, and those available to those skilled in the art. Constitutive promoter regulatory elements can also be used to direct continuous gene expression. Constitutive promoters include, but are not limited to, promoters from plant viruses, such as the 35S promoter of CaMV (Odell et al., Nature 313:810-812 (1985)), and promoters such as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)), ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)), pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)), MAS (Velten et al., EMBO J. 3:2723-2730 (1984)), maize H3 histone (Lepetit et al., Mol. Gen. Genetics [0015] The invention also provides a promoter for expressing genes (e.g., zein, oleosin, napin, ACP, globulin, etc.) in specific cell or tissue types, such as leaves or seeds, using tissue-specific promoter regulatory elements. Tissue-specific or tissue-preferred promoters include, but are not limited to, root-preferred promoters, such as the promoter from the phaseolin gene (Murai et al., Science 23:476-482 (1983), and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); leaf-specific and light-inducible promoters, such as the promoter from cab or ribulose bisphosphate carboxylase-oxygenase (rubisco) (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); anther-specific promoters, such as the promoter from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); pollen-specific promoters, such as the promoter from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)); or microspore-preferred promoters, such as the promoter from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)). Promoter regulatory elements can be activated at specific stages of plant development and plant tissues and organs, including but not limited to pollen-specific, embryo-specific, corn silk-specific, cotton fiber-specific, root-specific, and seed endosperm-specific promoter regulatory elements. Inducible promoter regulatory elements can be used, which respond to specific signals such as physical stimuli (heat shock genes); light (RUBP carboxylase); hormones (Em); metabolites; chemicals and stress to cause gene expression. Inducible promoters include, but are not limited to, a promoter from the ACEI system that responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); a promoter from the maize In2 gene that responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)), a promoter from the Tn10 Tet repressor (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)); and a promoter from a steroid hormone gene whose transcriptional activity is induced by glucocorticoids (Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

A signal sequence may be used to direct the polypeptide to an organelle or subcellular fraction or to the apoplast for secretion. See, e.g., Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al., Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, et al., Cell 39:499-509 (1984), and Steifel et al., Plant Cell 2:785-793 (1990). Such targeting sequences allow the desired protein to be translocated to the cellular structure where it functions most efficiently, or to the region of the cell where the cellular processes required for the desired phenotypic function are most concentrated.

In some embodiments, a signal sequence is used to direct the proteins of the invention to subcellular compartments, such as plastids or chloroplasts. Gene products, including heterologous gene products, can be targeted to plastids or chloroplasts by fusing the gene product to a signal sequence that is cleaved upon chloroplast import, thereby producing the mature protein. See, for example, Comai et al., J. Biol. Chem. 263:15104-15109 (1988), and van den Broeck et al., Nature 313:358-363 (1985). DNA encoding a suitable signal sequence can be isolated from cDNA encoding RUBISCO protein, CAB protein, EPSP synthase, GS2 protein, or from any naturally occurring chloroplast-targeting protein that contains a signal sequence (also known as a chloroplast transit peptide (CTP)) that directs the target protein to the chloroplast. Such chloroplast-targeting proteins are well known in the art. These chloroplast-targeting proteins are synthesized as larger precursor proteins containing an amino-terminal CTP that directs the precursor to the chloroplast import machinery. Specific endonucleases within the chloroplast typically cleave the CTP, thereby releasing the targeted mature protein, including active proteins such as enzymes, from the precursor into the chloroplast environment. Examples of coding sequences for peptides suitable for targeting genes or gene products to chloroplasts or plastids include the petunia EPSPS CTP, the Arabidopsis EPSPS CTP2 and introns, and other sequences known in the art. Specific examples of CTPs include, but are not limited to, the Arabidopsis ribulose bisphosphate carboxylase small subunit ats1A transit peptide, the Arabidopsis EPSPS transit peptide, and the maize ribulose bisphosphate carboxylase small subunit transit peptide. For a description of optimized transit peptides, see, e.g., Van den Broeck et al., Nature 313:358-363 (1985). Prokaryotic and eukaryotic signal peptide sequences are described, for example, in Michaelis et al., Ann. Rev. Microbiol. 36:425 (1982). Other examples of transit peptides that can be used in the present invention include chloroplast transit peptides, see Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126 (1991); Mazur et al., Plant Physiol. 85:1110 (1987); Vorst et al., Gene 65:59 (1988); Chen and Jagendorf, J. Biol. Chem. 268:2363-2367 (1993); the transit peptide from the rbcS gene of tobacco (Nicotiana plumbaginifolia) (Poulsen et al., Mol. Gen. Genet. 205:193-200 (1986)); and the transit peptide from Brassica napus acyl ACP thioesterase (Loader et al., Plant Mol. Biol. 23:769-778 (1993); Loader et al., Plant Physiol. 85:1110 (1987); Vorst et al., Gene 65:59 (1988); Chen and Jagendorf, J. Biol. Chem. 268:2363-2367 (1993); Physiol. 110:336-336(1995).

The genetically modified plants of the present invention can also be modified to delete or inactivate endogenous fatty acid synthase to reduce competition with exogenous PUFA synthase systems for malonyl-CoA, thereby increasing malonyl-CoA levels, and combinations thereof. See, for example, U.S. Application Publication No. 2007/0245431.

Genetically modified plants can be cultured in fermentation media or grown in a suitable culture medium, such as soil. Culture media suitable for higher plants include any plant growth medium, such as, but not limited to, soil, sand, any other specialized culture medium that supports root growth (such as vermiculite, perlite, etc.), or hydroponics, as well as suitable light, water, and nutritional supplements to optimize the growth of higher plants. PUFAs can be recovered from genetically modified plants by purification processes that extract the compounds from the plants. PUFAs can be recovered by harvesting the plants and harvesting the oil from the plants (from oilseeds). The plants can be consumed in their natural state or further processed into consumable products. In some embodiments, the present invention relates to genetically modified plants, wherein as a result of the genetic modification, the plant produces at least one PUFA, wherein as a result of the genetic modification of the plant, the total fatty acid profile of the plant or the part of the plant that accumulates the PUFA contains a detectable amount of PUFAs. In some embodiments, the plant is an oilseed plant. In some embodiments, the oilseed plant produces PUFAs in its mature seeds or contains PUFAs in the oil of its seeds.

Various mammalian cell culture systems can also be used to express recombinant proteins. The expression vector contains a replication origin, a suitable promoter and enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences.

Heterologous expression method

The present invention relates to a method for producing at least one PUFA, comprising expressing a PUFA synthase system in a host cell under conditions effective to produce the PUFA, wherein the PUFA synthase system comprises any of the isolated and recombinant nucleic acid molecules described herein, and combinations thereof, wherein at least one PUFA is produced. In some embodiments, the at least one PUFA comprises DHA, EPA, or a combination thereof. In some embodiments, the host cell is a plant cell, an isolated animal cell, or a microbial cell. In some embodiments, the host cell is a thraustochytrid.

The present invention relates to a method for producing lipids enriched in DHA, EPA, or a combination thereof, comprising expressing a PUFA synthase gene in a host cell under conditions effective to produce the lipids, wherein the PUFA synthase gene in the host cell comprises any of the isolated nucleic acid molecules and recombinant nucleic acid molecules described herein, and combinations thereof, wherein lipids enriched in DHA, EPA, or a combination thereof are produced.

The present invention relates to a method for isolating lipids from a host cell, comprising expressing a PUFA synthase gene in the host cell under conditions effective to produce lipids, and isolating lipids from the host cell, wherein the PUFA synthase system of the host cell comprises any of the isolated nucleic acid molecules and recombinant nucleic acid molecules described herein, and combinations thereof.

In some embodiments, one or more lipid components containing PUFAs are isolated from the host cells. In some embodiments, the one or more components isolated from the host cells include a total fatty acid component, a sterol ester component, a triglyceride component, a free fatty acid component, a sterol component, a diglycerol component, a phospholipid component, or a combination thereof. In some embodiments, the PUFAs isolated from the host cells are enriched in ω-3 fatty acids, ω-6 fatty acids, or a combination thereof, depending on the composition of the PUFA synthase system introduced into the host cells. In some embodiments, the PUFAs are enriched in DHA, EPA, DPA n-6, ARA, or a combination thereof, depending on the composition of the PUFA synthase system introduced into the host cells. In some embodiments, the PUFAs are enriched in DHA, EPA, or a combination thereof. In some embodiments, the PUFA profile of the PUFAs isolated from the host cells includes a high concentration of DHA and a low concentration of EPA, ARA, DPA n-6, or a combination thereof. In some embodiments, the PUFA profile of the PUFAs isolated from the host cells includes a high concentration of DHA and EPA and a low concentration of ARA, DPA n-6, or a combination thereof. In some embodiments, the PUFA profile of the PUFAs isolated from the host cells includes a high concentration of EPA and a low concentration of DHA, ARA, DPAn-6, or a combination thereof.

The present invention relates to methods for replacing an inactivated or deleted PUFA synthase activity, introducing a new PUFA synthase activity, or enhancing an existing PUFA synthase activity in an organism having PUFA synthase activity, comprising expressing any of the isolated and recombinant nucleic acid molecules described herein, and combinations thereof, in the organism under conditions effective to express the PUFA synthase activity. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences of a PF A1, PF A2, or PF A3 PUFA synthase described herein, encoding one or more PUFA synthase domains. In some embodiments, the PUFA profile of the organism is altered following introduction of one or more nucleic acid molecules of the present invention. In some embodiments, the altered PUFA profile comprises an increase in ω-3 fatty acids and a decrease in ω-6 fatty acids. In some embodiments, the altered PUFA profile comprises an increase in ω-6 fatty acids and a decrease in ω-3 fatty acids. In some embodiments, both ω-3 and ω-6 fatty acids are increased. In some embodiments, the amount of DHA is increased, while the amount of one or more of EPA, ARA, DPA n-6, or a combination thereof is maintained or decreased. In some embodiments, the amounts of EPA and DHA are increased, while the amounts of ARA, DPA n-6, or a combination thereof are maintained or decreased. In some embodiments, the amount of EPA is increased, while the amounts of one or more of EPA, ARA, DPA n-6, or a combination thereof are maintained or decreased. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of PFA3 or one or more domains thereof. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of PFA3 or one or more domains thereof, and the amount of omega-3 fatty acids in the organism is increased and the amount of omega-6 fatty acids is decreased. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of PFA2 or one or more domains thereof, and the amount of DHA in the organism is increased and the amount of EPA is decreased.

The present invention relates to a method for increasing the production of DHA, EPA, or a combination thereof in an organism having PUFA synthase activity, comprising expressing any of the isolated nucleic acid molecules and recombinant nucleic acid molecules described herein, and combinations thereof, in an organism under conditions effective to produce DHA, EPA, or a combination thereof, wherein the PUFA synthase activity in the organism replaces an inactive or deleted activity, introduces a new activity, or enhances an existing activity, and the production of DHA, EPA, or a combination thereof in the organism is increased.

Having summarized the present invention, a further understanding of the present invention can be obtained by referring to the examples provided herein. The following examples are for illustrative purposes only and are not intended to be limiting.

Example 1

Degenerate primers for the KS and DH PUFA synthase domains were designed and the corresponding sequences were isolated from an isolated microorganism deposited under ATCC Accession No. PTA-9695, also known as Schizochytrium ATCC PTA-9695.

Based on the published PFA1 (formerly known as orfA or ORF 1) sequences of Shewanella japonica, Schizochytrium sp. ATCC 20888, Thraustochytrium aureum (ATCC 34304), and Thraustochytrium sp. 23B ATCC 20892, degenerate primers for the KS region (i.e., the region containing the KS domain) of Schizochytrium sp. ATCC PTA-9695 PFA1 were designed:

prDS173 (forward): GATCTACTGCAAGCGCGGNGGNTTYAT (SEQ ID NO: 62), and

prDS174 (reverse): GGCGCAGGCGGCRTCNACNAC (SEQ ID NO: 63).

Based on the published sequences of Moritella marina, Schizochytrium sp. ATCC 20888, Photobacter profundum, and Thraustochytrium 23B ATCC 20892, degenerate primers for the DH region of Schizochytrium sp. ATCC PTA-9695 PFA3 (formerly known as orfC or ORF3) were designed:

JGM190 (forward): CAYTGGTAYTTYCCNTGYCAYTT (SEQ ID NO: 64); and

BLR242 (reverse): CCNGGCATNACNGGRTC (SEQ ID NO: 65).

The PCR conditions using chromosomal DNA template were as follows: 0.2 μM dNTP, 0.1 μM of each primer, 8% DMSO, 200 ng of chromosomal DNA, 2.5 U of Herculase II Fusion Polymerase (Stratagene), and 1× Herculase buffer (Stratagene), in a total volume of 50 μL. The PCR method included the following steps: (1) 98°C for 3 minutes; (2) 98°C for 30 seconds; (3) 50°C for 30 seconds; (4) 72°C for 2 minutes; (5) repeating steps 2-4 for 40 cycles; (6) 72°C for 5 minutes; and (7) storage at 6°C.

For the two pairs of primers, PCR was performed using a chromosomal template from Schizochytrium sp. ATCC Accession No. PTA-9695 to obtain different DNA products of the expected size. The PCR products were cloned into the vector pJET1.2/blunt end (Fermentas) according to the manufacturer's manual, and the insert sequence was determined using the provided standard primers.

The DNA sequences obtained from the PCR products were compared with known sequences from NCBI GenBank using a standard BLASTx search (BLASTx parameters: low complexity filtering on; matrix: BLOSUM62; gap depletion; presence 11, extension 1. Stephen F. Altschul, Thomas L. Madden, Alejandro A. Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.).

At the amino acid level, the highest levels of homology to the amino acid sequences deduced from cloned DNA containing the KS fragment of Thraustochytrium ATCC PTA-9695 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit A" (identity = 87%; positive rate = 92%); Shewanella oneidensis MR-1 "Multidomain β-ketoacyl synthase" (identity = 49%; positive rate = 64%); and Shewanella sp. MR-4 "β-ketoacyl synthase" (identity = 49%; positive rate = 64%).

At the amino acid level, the highest levels of homology to the amino acid sequences of cloned DNA containing the DH fragment of Thraustochytrium ATCC PTA-9695 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit C" (identity = 61%; positive rate = 71%); Shewanella pealeana ATCC 700345 "β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA/FabZ" (identity = 35%; positive rate = 50%); and Shewanella sediminis. HAW-EB3 "ω-3 polyunsaturated fatty acid synthase PfaC" (identity = 34%; positive rate = 50%).

Example 2

PUFA synthase genes were identified from Schizochytrium sp. ATCC PTA-9695.

Prepare genomic DNA from microorganisms using standard procedures. See, for example, Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Briefly: (1) Centrifuge 500 μL of cells from a mid-logarithmic culture. Centrifuge the cells again and remove any remaining liquid from the cell pellet using a small-bore pipette tip.

(2) The pellet was resuspended in 200 μL lysis buffer (20 mM Tris pH 8.0, 125 μg/mL proteinase K, 50 mM NaCl, 10 mM EDTA pH 8.0, 0.5% SDS);

(3) Lyse cells at 50°C for 1 hour;

(4) Use a pipette to transfer the lysis mixture into a 2 mL tube of phase lock gel (PLG-Eppendorf);

(5) Add an equal volume of P:C:I and mix for 1.5 hours;

(6) Centrifuge the tube at 12k x g for 5 minutes;

(7) Remove the aqueous phase from the gel in the PLG tube, add an equal volume of chloroform to the aqueous phase, and mix for 30 minutes;

(8) Centrifuge the tube at 14k x g for approximately 5 minutes;

(9) Aspirate the upper layer (aqueous phase) from the chloroform and place it in a new test tube;

(10) Add 0.1 volume of 3M NaOAC and mix (invert several times);

(11) Add 2 volumes of 100% EtOH and mix (invert several times). A genomic DNA precipitate is formed at this stage.

(12) Centrifuge the tube at 4°C (14kJ) for approximately 15 minutes in a microcentrifuge.

(13) Carefully pour off the liquid, leaving the genomic DNA at the bottom of the tube;

(14) Wash the pellet with 0.5 mL of 70% EtOH;

(15) Centrifuge the tube at 4°C (14kJ) for approximately 5 minutes in a microcentrifuge.

(16) Carefully pour off the EtOH and dry the genomic DNA pellet;

and (17) adding appropriate volumes of H ₂ O and RNase directly to the genomic DNA pellet.

Using the isolated genome, a recombinant library containing large fragments (approximately 40 kB) was generated in the cosmid pWEB-TNC ^™ (Epicentre) according to the manufacturer's manual. The cosmid library was screened using a ³² P radiolabeled probe according to standard colony hybridization procedures (Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The probe contained DNA homologous to the published PUFA synthase sequences from other organisms described in Example 1. These probes were generated by restriction enzyme digestion of cloned fragments from the pJET1.2/blunt-end clones described above and labeled using standard methods. In all cases, strong hybridization of each probe to certain cosmids indicated that the clones contained DNA homologous to the PUFA synthase gene.

Cosmid clone pDS115 showed strong binding to the probe for the KS region and was selected for DNA sequencing of the Schizochytrium ATCC PTA-9695 PFA1 gene. Cosmid clone pDS115, containing the Schizochytrium ATCC PTA-9695 PFA1 and PFA2 genes, was deposited with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, under the Budapest Treaty on January 27, 2009, under ATCC Accession No. PTA-9737. Sequencing primers for the KS region DNA sequence determined in Example 1 were designed using standard methods. To determine the DNA sequence of Schizochytrium sp. ATCC PTA-9695 PFA1, several rounds of DNA sequencing were performed, including design of subsequent sequencing primers using standard methods to "walk through" the cosmid clone.

In the previously disclosed thraustochytrid PUFA synthase system, the PUFA synthase genes PFA1 and PFA2 are clustered and transcribed in opposite directions. This is also true for PFA1 and PFA2 from Schizochytrium ATCC PTA-9695. By "walking" the DNA sequence from the cosmid clone pDS115, it was determined that the start site of PFA2 is conceptually 493 nucleotides from the start site of PFA1 and is transcribed in opposite directions. Every nucleotide base pair of the Schizochytrium ATCC PTA-9695 PFA1 and PFA2 PUFA synthase genes was covered by at least two independent high-quality DNA sequencing reactions, with a minimum pooled Phred score of 40 (99.99% confidence level).

Cosmid clone pBS4 showed strong binding to the probe in the DH region and was selected for DNA sequencing of the Schizochytrium ATCC PTA-9695 PFA3 gene. Cosmid clone pBS4, containing the Schizochytrium ATCC PTA-9695 PFA3 gene, was deposited under the Budapest Treaty with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, on January 27, 2009, under ATCC Accession No. PTA-9736. Sequencing primers for the DH region DNA sequence determined in Example 1 were designed using standard methods. To determine the DNA sequence of S. pombe ATCC PTA-9695 PFA3, several rounds of DNA sequencing were performed, including the design of subsequent sequencing primers using standard methods to "walk through" the cosmid clone. Every nucleotide base pair of the Schizochytrium sp. ATCC PTA-9695 PFA3 PUFA synthase gene was covered by at least two independent high-quality DNA sequencing reactions with a minimum pooled Phred score of 40 (99.99% confidence level).

Table 1 shows the identities of the polynucleotide sequences of Schizochytrium sp. ATCC PTA-9695 PFA1 (SEQ ID NO: 1), PFA2 (SEQ ID NO: 3), and PFA3 (SEQ ID NO: 5) compared to previously published sequences. Identities were determined using the scoring matrix "swgapdnamt" in the "AlignX" program of VectorNTI, a standard program for DNA alignment.

Table 1: Percentage identity of polynucleotide sequences of PFA1, PFA2 and PFA3

Table 2 shows the identity of the amino acid sequences of Schizochytrium sp. ATCC PTA-9695 Pfa1p (SEQ ID NO: 2), Pfa2p (SEQ ID NO: 4), and Pfa3p (SEQ ID NO: 6) to previously published PUFA synthase amino acid sequences. Identities were determined using the scoring matrix "blosum62mt2" in the "AlignX" program of VectorNTI, a standard protein alignment program.

Table 2: Percent identity of amino acid sequences of Pfa1p, Pfa2p and Pfa3p

Example 3

Domain analysis was performed to annotate the PUFA synthase domains and active site sequence coordinates of Schizochytrium sp. ATCC PTA-9695 PFA1, PFA2, and PFA3. Domains were identified based on homology to known PUFA synthase, fatty acid synthase, and polyketide synthase domains.

Table 3 shows the structural domains and active sites associated with Schizochytrium ATCC PTA-9695 PFA1.

Table 3: Structural analysis of the PFA1 domain of Schizochytrium ATCC PTA-9695

The first domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is the KS domain. The nucleotide sequence encoding the KS domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is set forth herein as SEQ ID NO:7, corresponding to positions 7-1401 of SEQ ID NO:1. The amino acid sequence encoding the KS domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is set forth herein as SEQ ID NO:8, corresponding to positions 3-467 of SEQ ID NO:2. The KS domain contains the active site motif: DXAC* (SEQ ID NO:43), where the * acyl binding site corresponds to C203 of SEQ ID NO:2. Likewise, there is a characteristic motif at the end of the KS domain: GFGG (SEQ ID NO: 44), corresponding to positions 455-458 of SEQ ID NO: 2 and positions 453-456 of SEQ ID NO: 8.

The second domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is the MAT domain. The nucleotide sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa1 MAT domain is set forth herein as SEQ ID NO:9, corresponding to positions 1798-2700 of SEQ ID NO:1. The amino acid sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa1 MAT domain is set forth herein as SEQ ID NO:10, corresponding to positions 600-900 of SEQ ID NO:2. The MAT domain contains the active site motif: GHS*XG (SEQ ID NO:46), where the * acyl binding site corresponds to S699 of SEQ ID NO:2.

Domains 3-8 of Schizochytrium sp. ATCC PTA-9695 Pfa1 consist of six tandem ACP domains, designated herein as ACP1, ACP2, ACP3, ACP4, ACP5, and ACP6. The nucleotide sequence comprising the first ACP domain, ACP1, is designated herein as SEQ ID NO:13 and is encompassed within the nucleotide sequence of approximately positions 3325-3600 of SEQ ID NO:1. The amino acid sequence comprising ACP1 is designated herein as SEQ ID NO:14 and is encompassed within the amino acid sequence of approximately positions 1109-1200 of SEQ ID NO:2. The nucleotide sequence comprising ACP2 is designated herein as SEQ ID NO:15 and is encompassed within the nucleotide sequence of approximately positions 3667-3942 of SEQ ID NO:1. The amino acid sequence comprising ACP2 is designated herein as SEQ ID NO:16 and is encompassed within the amino acid sequence of approximately positions 1223-1314 of SEQ ID NO:2. A nucleotide sequence comprising ACP3 is designated herein as SEQ ID NO: 17 and is encompassed within the nucleotide sequence of approximately positions 4015-4290 of SEQ ID NO: 1. An amino acid sequence comprising ACP3 is designated herein as SEQ ID NO: 18 and is encompassed within the amino acid sequence of approximately positions 1339-1430 of SEQ ID NO: 2. A nucleotide sequence comprising ACP4 is designated herein as SEQ ID NO: 19 and is encompassed within the nucleotide sequence of approximately positions 4363-4638 of SEQ ID NO: 1. An amino acid sequence comprising ACP4 is designated herein as SEQ ID NO: 20 and is encompassed within the amino acid sequence of approximately positions 1455-1546 of SEQ ID NO: 2. A nucleotide sequence comprising ACP5 is designated herein as SEQ ID NO: 21 and is encompassed within the nucleotide sequence of approximately positions 4711-4986 of SEQ ID NO: 1. The amino acid sequence comprising ACP5 is designated herein as SEQ ID NO:22 and is encompassed within the amino acid sequence of approximately positions 1571-1662 of SEQ ID NO:2. The nucleotide sequence comprising ACP6 is designated herein as SEQ ID NO:23 and is encompassed within the nucleotide sequence of approximately positions 5053-5328 of SEQ ID NO:1. The amino acid sequence comprising ACP6 is designated herein as SEQ ID NO:24 and is encompassed within the amino acid sequence of approximately positions 1685-1776 of SEQ ID NO:2. Together, all six ACP domains cover the region of approximately positions 3298-5400 of SEQ ID NO:1 of Schizochytrium sp. ATCC PTA-9695 Pfa1, corresponding approximately to amino acid positions 1100-1800 of SEQ ID NO:2. The nucleotide sequence of the entire ACP region, encompassing all six domains, is herein designated as SEQ ID NO: 11; the amino acid sequence of the entire ACP region, encompassing all six domains, is designated as SEQ ID NO: 12. The six ACP regions within SEQ ID NO: 11 are repeated approximately every 342 nucleotides (the actual number of amino acids between adjacent active site serines ranges from 114 to 116 amino acids). Each of the six ACP domains contains a pantetheine-binding motif, LGIDS* (SEQ ID NO: 47), where S* is the pantetheine-binding site serine (S). The pantetheine-binding site serine (S) is located near the center of each ACP domain sequence. The positions of the active site serines of the six ACPD domains (i.e., the pantetheine binding site) for the amino acid sequence of SEQ ID NO: 2 are: ACP1 = S1152, ACP2 = S1266, ACP3 = S1382, ACP4 = S1498, ACP5 = S1614, and ACP6 = S1728.

The ninth domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is the KR domain. The nucleotide sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa KR domain is shown herein as SEQ ID NO:25, corresponding to positions 5623-7800 of SEQ ID NO:1. The amino acid sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa1 KR domain is shown herein as SEQ ID NO:26, corresponding to positions 1875-2600 of SEQ ID NO:2. Within the KR domain is a core region (contained in the nucleotide sequence of SEQ ID NO:48 and the amino acid sequence of SEQ ID NO:49) homologous to short-chain aldehyde dehydrogenases (of which KR is a member).

This core region spans approximately positions 5998-6900 of SEQ ID NO:1, corresponding to amino acid positions 2000-2300 of SEQ ID NO:2. The tenth domain of Schizochytrium sp. ATCC PTA-9695 Pfa1 is the DH domain. The nucleotide sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa1 DH domain is shown herein as SEQ ID NO:27, corresponding to positions 7027-7065 of SEQ ID NO:1. The amino acid sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa1 DH domain is shown herein as SEQ ID NO:28, corresponding to positions 2343-2355 of SEQ ID NO:2. The DH domain contains a conserved active site motif (see Donadio, S. and Katz., L., Gene 111(1):51-60 (1992)): LxxHxxxGxxxxP (SEQ ID NO:50).

Table 4 shows the structural domains and active sites associated with Schizochytrium sp. ATCC PTA-9695 PFA2.

Table 4: Analysis of the PFA2 domain of Schizochytrium ATCC PTA-9695

The first domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is the KS domain. The nucleotide sequence encoding the KS domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is set forth herein as SEQ ID NO:29, corresponding to positions 10-1350 of SEQ ID NO:3. The amino acid sequence encoding the KS domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is set forth herein as SEQ ID NO:30, corresponding to positions 4-450 of SEQ ID NO:4. The KS domain contains the active site motif: DXAC* (SEQ ID NO:43), where the * acyl binding site corresponds to C191 of SEQ ID NO:4. Likewise, there is a characteristic motif at the end of the KS domain: GFGG (SEQ ID NO: 44), corresponding to positions 438-441 of SEQ ID NO: 4 and positions 435-438 of SEQ ID NO: 30.

The third domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is the CLF domain. The nucleotide sequence encoding the CLF domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is shown herein as SEQ ID NO:31, corresponding to positions 1408-2700 of SEQ ID NO:3. The amino acid sequence encoding the CLF domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is shown herein as SEQ ID NO:32, corresponding to positions 470-900 of SEQ ID NO:4.

The third domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is the AT domain. The nucleotide sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa2 AT domain is set forth herein as SEQ ID NO:33, corresponding to positions 2998-4200 of SEQ ID NO:3. The amino acid sequence encoding the Schizochytrium sp. ATCC PTA-9695 Pfa2 AT domain is set forth herein as SEQ ID NO:34, corresponding to positions 1000-1400 of SEQ ID NO:4. The AT domain contains the active site motif GxS*xG (SEQ ID NO:52), which is characteristic of acyltransferase (AT) proteins. The active site serine residue corresponds to S1141 of SEQ ID NO:4.

The fourth domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is the ER domain. The nucleotide sequence encoding the ER domain of Schizochytrium sp. ATCC PTA-9695 Pfa2 is shown herein as SEQ ID NO:35, corresponding to positions 4498-5700 of SEQ ID NO:3. The amino acid sequence encoding the Pfa2 ER domain is shown herein as SEQ ID NO:36, corresponding to positions 1500-1900 of SEQ ID NO:4.

Table 5 shows the structural domains and active sites associated with Schizochytrium sp. ATCC PTA-9695 PFA3.

Table 5: Structural analysis of the PFA3 domain of Schizochytrium ATCC PTA-9695

The first and second domains of Schizochytrium sp. ATCC PTA-9695 Pfa3 are referred to herein as DH1 and DH2, respectively. The nucleotide sequence encoding the DH1 domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is set forth herein as SEQ ID NO:37, corresponding to positions 1-1350 of SEQ ID NO:5. The amino acid sequence encoding the DH1 domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is set forth herein as SEQ ID NO:38, corresponding to positions 1-450 of SEQ ID NO:6. The nucleotide sequence encoding the DH2 domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is set forth herein as SEQ ID NO:39, corresponding to positions 1501-2700 of SEQ ID NO:5. The amino acid sequence comprising the DH2 domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is set forth herein as SEQ ID NO:40, corresponding to positions 501-900 of SEQ ID NO:6. The DH domain comprises the active site motif: FxxH*F (SEQ ID NO:53). The nucleotide sequence comprising the active site motif in DH1 corresponds to positions 931-933 of SEQ ID NO:5, while the nucleotide sequence comprising the active site motif in DH2 corresponds to positions 2401-2403 of SEQ ID NO:5. The active site H* in the motif FxxH*F is based on data from Leesong et al., Structure 4:253-64 (1996) and Kimber et al., J Biol Chem. 279:52593-602 (2004). The active site H* in DH1 corresponds to H310 of SEQ ID NO: 6, and the active site H* in DH2 corresponds to H801 of SEQ ID NO: 6.

The third domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is the ER domain. The nucleotide sequence encoding the ER domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is shown herein as SEQ ID NO:41, corresponding to positions 2848-4200 of SEQ ID NO:5. The amino acid sequence encoding the ER domain of Schizochytrium sp. ATCC PTA-9695 Pfa3 is shown herein as SEQ ID NO:42, corresponding to positions 950-1400 of SEQ ID NO:6.

Example 4

Degenerate primers for the KS, ER, and DH PUFA synthase domains were designed and the corresponding sequences were isolated from an isolated microorganism deposited under ATCC Accession No. PTA-10212, also known as Thraustochytrium sp. ATCC PTA-10212.

Based on the published PFA1 (formerly known as orfA or ORF 1) sequences of Schizochytrium sp. ATCC 20888, Thraustochytrium aureum (ATCC 34304), and Thraustochytrium sp. 23B ATCC 20892, degenerate primers for the KS region (i.e., the region containing the KS domain) of Thraustochytrium sp. ATCC PTA-10212 PFA1 were designed:

prDS233 (forward): TGATATGGGAGGAATGAATTGTGTNGTNGAYGC (SEQ ID NO: 123)

prDS235 (reverse): TTCCATAACAAAATGATAATTAGCTCCNCCRAANCC (SEQ ID NO: 124).

Based on the published PFA2 (formerly known as orfB or ORF 2) sequences of Shewanella japonicum, Schizochytrium sp. ATCC 20888, Thraustochytrium aureum (ATCC 34304), and Thraustochytrium sp. 23B ATCC 20892, degenerate primers for the ER region (i.e., the region containing the ER domain) of Thraustochytrium sp. ATCC PTA-10212 PFA2 were designed:

prDS183 (forward): GGCGGCCACACCGAYAAYMGNCC (SEQ ID NO: 125)

prDS184 (reverse): CGGGGCCGCACCANAYYTGRTA (SEQ ID NO: 126).

Based on the published PFA3 (formerly known as orfC or ORF 3) sequences of Shewanella japonicum, Schizochytrium sp. ATCC 20888, Thraustochytrium aureum (ATCC 34304), and Thraustochytrium sp. 23B ATCC 20892, degenerate primers for the ER region (i.e., the region containing the ER domain) of Thraustochytrium sp. ATCC PTA-10212 PFA3 were designed:

prDS181 (forward): TCCTTCGGNGCNGSNGG (SEQ ID NO: 127)

prDS184 (reverse): CGGGGCCGCACCANAYYTGRTA (SEQ ID NO: 126).

The DH region of PFA3 from Thraustochytrium ATCC PTA-10212 was amplified using the degenerate primers JGM190 (forward, SEQ ID NO: 64) and BLR242 (reverse, SEQ ID NO: 65) as described above.

The PCR conditions using chromosomal DNA template were as follows: 0.2 μM dNTP, 0.1 μM of each primer, 6% DMSO, 200 ng of chromosomal DNA, 2.5 U of Herculase II Fusion Polymerase (Schwagen), and 1X Herculase buffer (Schwagen), in a total volume of 50 μL. The PCR method included the following steps: (1) 98°C for 3 minutes; (2) 98°C for 30 seconds; (3) 54°C for 45 seconds; (4) 72°C for 2 minutes; (5) repeating steps 2-4 for 40 cycles; (6) 72°C for 5 minutes; and (7) storage at 6°C.

For all primer pairs, PCR yielded DNA products of the expected size using a chromosomal template from Thraustochytrium ATCC PTA-10212. PCR products were cloned into the pJET1.2/blunt-end vector (EnzymeTex) according to the manufacturer's instructions, and the insert sequence was determined using the provided standard primers.

The DNA sequences obtained from the PCR products were compared with known sequences from NCBI GenBank as described in Example 1.

At the amino acid level, the sequences with the highest levels of homology to the amino acid sequence deduced from cloned DNA containing the KS fragment of PFA1 from Thraustochytrium ATCC PTA-10212 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit A" (identity = 80%; positive rate = 90%); Shewanella benthica KT99 "ω-3 polyunsaturated fatty acid synthase PfaA" (identity = 51%; positive rate = 67%); Shewanella loihica PV-4 "β-ketoacyl synthase" (identity = 50%; positive rate = 67%); Shewanella woodyi ATCC 51908 "Polyketide-type polyunsaturated fatty acid synthase PfaA" (identity = 51%; positive rate = 66%).

At the amino acid level, the sequences with the highest levels of homology to the amino acid sequence deduced from cloned DNA containing the ER fragment of PFA2 from Thraustochytrium sp. ATCC PTA-10212 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit B" (identity = 70%; positive rate = 85%); Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit C" (identity = 66%; positive rate = 83%); Nodularia spumigena CCY9414 "2-nitropropane dioxygenase" (identity = 57%; positive rate = 74%); and Moritella sp. PE36 "Polyunsaturated fatty acid synthase PfaD" (identity = 57%; positive rate = 71%).

At the amino acid level, the sequences with the highest levels of homology to the amino acid sequence deduced from cloned DNA of the ER fragment containing PFA3 from Thraustochytrium ATCC PTA-10212 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit C" (identity = 80%; positive rate = 90%); Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit B" (identity = 78%; positive rate = 89%); Antarctic bacterium PE36 "Polyunsaturated fatty acid synthase PfaD" (identity = 56%; positive rate = 71%); Shewanella amazonensis SB2B "ω-3 polyunsaturated fatty acid synthase" (identity = 55%; positive rate = 73%).

At the amino acid level, the sequences with the highest levels of homology to the amino acid sequence deduced from cloned DNA containing the DH fragment of PFA3 from Thraustochytrium ATCC PTA-10212 were: Schizochytrium ATCC 20888 "Polyunsaturated fatty acid synthase subunit C" (identity = 63%; positive rate = 76%); Shewanella pealeana ATCC 700345 "β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA/FabZ" (identity = 35%; positive rate = 53%); Shewanella piezotolerans WP3 "Multidomain β-ketoacyl synthase" (identity = 36%; positive rate = 52%); Shewanella benthica KT99 "ω-3 polyunsaturated fatty acid synthase PfaC" (identity = 35%; positive rate = 51%).

Example 5

PUFA synthase genes were identified from Schizochytrium sp. ATCC PTA-10212.

Thaw 1 mL of cells from a -80°C cryovial at room temperature and add them to a 250 mL unbaffled culture flask containing 50 mL of liquid HSFM medium (described below). Incubate the flask at 23°C for 3 days. Collect the cells and use them for standard cell artificial chromosome (BAC) library construction (Lucigen Corporation, Middleton, WI USA).

Table 6: HSFM culture medium

Nitrogen feed:

Typical culture conditions include:

A recombinant BAC library containing large fragments (approximately 120 kB) was processed according to the manufacturer's instructions for the BAC vector pSMART (Lucigen Corporation). The BAC library was screened using a standard colony hybridization procedure using a ^32P radiolabeled probe (Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The probe contained DNA homologous to the published PUFA synthase sequences from other organisms described in Example 4. These probes were generated by DNA restriction enzyme digestion of cloned fragments from the pJET1.2/blunt-end clones described above and labeled using standard methods. In all cases, strong hybridization of each probe to certain BACs indicated that the clone contained DNA homologous to the PUFA synthase gene.

The BAC clone pLR130 (also known as LuMaBAC 2M23) showed strong hybridization with probes in the KS and ER regions, indicating that it contained the PFA1 and PFA2 genes, and this clone was selected for DNA sequencing of the PFA1 and PFA2 genes of Thraustochytrium ATCC PTA-10212. BACs were sequenced by standard procedures (Eurofins MWG Operon, Huntsville, AL). The BAC clone pLR130, comprising the PFA1 and PFA2 genes, was deposited with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, under the Budapest Treaty on December 1, 2009, under ATCC Accession No. PTA-10511.

In previously published thraustochytrium PUFA synthase systems, the PUFA synthase genes PFA1 and PFA2 are clustered and transcribed in opposite directions. This is also true for PFA1 and PFA2 from Thraustochytrium sp. ATCC PTA-10212. Conceptually, the start site of PFA2 is located 693 nucleotides from the start site of PFA1 and is transcribed in opposite directions.

The BAC clone pDS127 (also known as LuMaBAC 9K17) showed strong hybridization with probes for the DH and ER regions of PFA3 and was selected for DNA sequencing of the PFA3 gene. The BAC clone pDS130, containing the PFA3 gene, was deposited under the Budapest Treaty on December 1, 2009, with the American Type Culture Collection, Patent Collection, 10801 University Avenue, Manassas, VA 20110-2209, under ATCC Accession No. PTA-10510. Sequencing primers were designed using standard methods for the DH and ER regions and DNA sequences determined in Example 4. To determine the DNA sequence of the Thraustochytrium ATCC PTA-10212 PFA3 gene, several rounds of DNA sequencing were performed, including the design of subsequent sequencing primers using standard methods, to "walk through" the BAC clones. Every nucleotide base pair of the PFA3 gene was covered by at least two independent high-quality DNA sequencing reactions, with a minimum pooled Phred score of 40 (99.99% confidence level).

Table 7 shows the identities of the Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 68), PFA2 (SEQ ID NO: 70), and PFA3 (SEQ ID NO: 72) polynucleotide sequences compared to previously published sequences and the sequence of Schizochytrium sp. PTA-9695. Identities were determined using the scoring matrix "swgapdnamt" in the "AlignX" program of VectorNTI, a standard program for DNA alignment.

Table 7: Percent identity of polynucleotide sequences of PFA1, PFA2 and PFA3

Table 8 shows the identity of the amino acid sequences of Thraustochytrium sp. ATCC PTA-10212 Pfa1p (SEQ ID NO: 69), Pfa2p (SEQ ID NO: 71), and Pfa3p (SEQ ID NO: 73) compared to previously published sequences and the amino acid sequence of the PUFA synthase from Schizochytrium sp. PTA-9695. Identities were determined using the scoring matrix "blosum62mt2" in the "AlignX" program of VectorNTI, a standard program for protein alignment.

Table 8: Percentage identity of amino acid sequences of Pfa1p, Pfa2p and Pfa3p

Example 6

Domain analysis was performed to annotate the PUFA synthase domains of Thraustochytrium sp. ATCC PTA-10212 PFA1, PFA2, and PFA3, as well as the sequence coordinates of their respective active sites. Domains were identified based on homology to known PUFA synthase, fatty acid synthase, and polyketide synthase domains.

Table 9 shows the structural domains and active sites associated with Thraustochytrium ATCC PTA-10212 PFA1.

Table 9: Thraustochytrid ATCC PTA-10212 PFA1 domain analysis

The first domain of Thraustochytrium ATCC PTA-10212 Pfa1 is the KS domain. The nucleotide sequence encoding the KS domain of Thraustochytrium ATCC PTA-10212 Pfa1 is set forth herein as SEQ ID NO:74, corresponding to positions 13-1362 of SEQ ID NO:68. The amino acid sequence encoding the KS domain of Thraustochytrium ATCC PTA-10212 Pfa1 is set forth herein as SEQ ID NO:75, corresponding to positions 5-454 of SEQ ID NO:69. The KS domain contains the active site motif: DXAC* (SEQ ID NO:43), where the * acyl binding site corresponds to C204 of SEQ ID NO:69. Similarly, a characteristic motif: GFGG (SEQ ID NO:44) is present at the terminus of the KS domain, corresponding to positions 451-454 of SEQ ID NO:69 and positions 447-450 of SEQ ID NO:75.

The second domain of Thraustochytrium ATCC PTA-10212 Pfa1 is the MAT domain. The nucleotide sequence encoding the MAT domain of Thraustochytrium ATCC PTA-10212 Pfa1 is set forth herein as SEQ ID NO:76, corresponding to positions 1783-2703 of SEQ ID NO:68. The amino acid sequence encoding the MAT domain of Thraustochytrium ATCC PTA-10212 Pfa1 is set forth herein as SEQ ID NO:77, corresponding to positions 595-901 of SEQ ID NO:69. The MAT domain comprises the active site motif: GHS*XG (SEQ ID NO:46), where the *acyl binding site corresponds to S695 of SEQ ID NO:69.

Domains 3-12 of Thraustochytrium ATCC PTA-10212 Pfa1p consist of ten tandem ACP domains, designated herein as ACP1, ACP2, ACP3, ACP4, ACP5, ACP6, ACP7, ACP8, ACP9, and ACP10. The nucleotide sequence comprising the first ACP domain, ACP1, is designated herein as SEQ ID NO:80 and is encompassed within the nucleotide sequence of approximately positions 3280-3534 of SEQ ID NO:68. The amino acid sequence comprising ACP1 is designated herein as SEQ ID NO:81 and is encompassed within the amino acid sequence of approximately positions 1094-1178 of SEQ ID NO:69. The nucleotide sequence comprising ACP2 is designated herein as SEQ ID NO:82 and is encompassed within the nucleotide sequence of approximately positions 3607-3861 of SEQ ID NO:68. An amino acid sequence comprising ACP2 is designated herein as SEQ ID NO:83 and is encompassed within the amino acid sequence of approximately positions 1203-1287 of SEQ ID NO:69. A nucleotide sequence comprising ACP3 is designated herein as SEQ ID NO:84 and is encompassed within the amino acid sequence of approximately positions 3934-4185 of SEQ ID NO:68. An amino acid sequence comprising ACP3 is designated herein as SEQ ID NO:85 and is encompassed within the amino acid sequence of approximately positions 1312-1396 of SEQ ID NO:69. A nucleotide sequence comprising ACP4 is designated herein as SEQ ID NO:86 and is encompassed within the amino acid sequence of approximately positions 4261-4515 of SEQ ID NO:68. An amino acid sequence comprising ACP4 is designated herein as SEQ ID NO:87 and is encompassed within the amino acid sequence of approximately positions 1421-1505 of SEQ ID NO:69. A nucleotide sequence comprising ACP5 is designated herein as SEQ ID NO:88 and is encompassed within the nucleotide sequence of approximately positions 4589-4842 of SEQ ID NO:68. An amino acid sequence comprising ACP5 is designated herein as SEQ ID NO:89 and is encompassed within the amino acid sequence of approximately positions 1530-1614 of SEQ ID NO:69. A nucleotide sequence comprising ACP6 is designated herein as SEQ ID NO:90 and is encompassed within the nucleotide sequence of approximately positions 4915-5169 of SEQ ID NO:68. An amino acid sequence comprising ACP6 is designated herein as SEQ ID NO:91 and is encompassed within the amino acid sequence of approximately positions 1639-1723 of SEQ ID NO:69. A nucleotide sequence comprising ACP7 is designated herein as SEQ ID NO:92 and is encompassed within the nucleotide sequence of approximately positions 5242-5496 of SEQ ID NO:68. An amino acid sequence comprising ACP7 is designated herein as SEQ ID NO:93 and is encompassed within the amino acid sequence of approximately positions 1748-1832 of SEQ ID NO:69. A nucleotide sequence comprising ACP8 is designated herein as SEQ ID NO:94 and is encompassed within the amino acid sequence of approximately positions 5569-5832 of SEQ ID NO:68. An amino acid sequence comprising ACP8 is designated herein as SEQ ID NO:95 and is encompassed within the amino acid sequence of approximately positions 1857-1941 of SEQ ID NO:69. A nucleotide sequence comprising ACP9 is designated herein as SEQ ID NO:96 and is encompassed within the amino acid sequence of approximately positions 5896-6150 of SEQ ID NO:68. An amino acid sequence comprising ACP9 is designated herein as SEQ ID NO:97 and is encompassed within the amino acid sequence of approximately positions 1966-2050 of SEQ ID NO:69. The nucleotide sequence comprising ACP10 is designated herein as SEQ ID NO:98 and is encompassed within the nucleotide sequence of approximately positions 6199-6453 of SEQ ID NO:68. The amino acid sequence comprising ACP10 is designated herein as SEQ ID NO:99 and is encompassed within the amino acid sequence of approximately positions 2067-2151 of SEQ ID NO:69. All ten ACP domains together cover the region of approximately positions 3208-6510 of SEQ ID NO:68 of Thraustochytrium ATCC PTA-10212 Pfa1, corresponding to amino acid positions 1070-2170 of SEQ ID NO:69. The nucleotide sequence of the entire ACP region, encompassing all ten domains, is designated herein as SEQ ID NO:78, and the amino acid sequence of the entire ACP region, encompassing all six domains, is designated herein as SEQ ID NO:79. The 10 ACP domains within SEQ ID NO:78 are repeated approximately every 327 nucleotides (the actual number of amino acids between adjacent active site serines ranges from 101 to 109 amino acids). Each of the 10 ACP domains contains a pantetheine binding motif LGIDS* (SEQ ID NO:47), where S* is the pantetheine binding site serine (S). The pantetheine binding site serine (S) is located near the center of each ACP domain sequence. The positions of the active site serines of the six ACPD domains (i.e., the pantetheine binding site) for the amino acid sequence of SEQ ID NO: 69 are: ACP1 = S1135, ACP2 = S1244, ACP3 = S1353, ACP4 = S1462, ACP5 = S1571, ACP6 = S1680, APC7 = S1789, ACP8 = S1898, ACP9 = S2007, and ACP10 = S2108.

The 13th domain of Thraustochytrium ATCC PTA-10212 Pfa1 is the KR domain. The nucleotide sequence encoding the Pfa1 KR domain is set forth herein as SEQ ID NO:100, corresponding to positions 6808-8958 of SEQ ID NO:68. The amino acid sequence encoding the Pfa1 KR domain is set forth herein as SEQ ID NO:101, corresponding to positions 2270-2986 of SEQ ID NO:69. Within the KR domain is a core region (enclosed in the nucleotide sequence of SEQ ID NO:116 and the amino acid sequence of SEQ ID NO:117) homologous to short-chain aldehyde dehydrogenases (of which KR is a member). This core region extends from approximately positions 5998-6900 of SEQ ID NO:68, corresponding to amino acid positions 2000-2300 of SEQ ID NO:69.

The 14th domain of Thraustochytrium ATCC PTA-10212 Pfa1 is the DH domain. The nucleotide sequence encoding the Pfa1 DH domain is shown herein as SEQ ID NO: 118, corresponding to positions 7027-7065 of SEQ ID NO: 68. The amino acid sequence encoding the Pfa1 DH domain is shown herein as SEQ ID NO: 119, corresponding to positions 2343-2355 of SEQ ID NO: 69. The DH domain contains a conserved active site motif (see Donadio, S. and Katz., L., Gene 111(1): 51-60 (1992)): LxxHxxxGxxxxP (SEQ ID NO: 50).

Table 10 shows the structural domains and active sites associated with Thraustochytrium ATCC PTA-10212 PFA2.

Table 10: Thraustochytrid ATCC PTA-10212 PFA2 domain analysis

The first domain of Thraustochytrium ATCC PTA-10212 Pfa2 is the KS domain. The nucleotide sequence encoding the KS domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:102, corresponding to positions 10-1320 of SEQ ID NO:70. The amino acid sequence encoding the KS domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:103, corresponding to positions 4-440 of SEQ ID NO:71. The KS domain contains the active site motif: DXAC* (SEQ ID NO:43), where the * acyl binding site corresponds to C191 of SEQ ID NO:71. Similarly, a characteristic motif: GFGG (SEQ ID NO:44) is present at the terminus of the KS domain, corresponding to positions 423-426 of SEQ ID NO:71 and positions 1267-1278 of SEQ ID NO:70.

The second domain of Thraustochytrium ATCC PTA-10212 Pfa2 is the CLF domain. The nucleotide sequence encoding the CLF domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:104, corresponding to positions 1378-2700 of SEQ ID NO:70. The amino acid sequence encoding the CLF domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:105, corresponding to positions 460-900 of SEQ ID NO:71.

The third domain of Thraustochytrium ATCC PTA-10212 Pfa2 is the AT domain. The nucleotide sequence encoding the AT domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:106, corresponding to positions 2848-4200 of SEQ ID NO:70. The amino acid sequence encoding the AT domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:107, corresponding to positions 950-1400 of SEQ ID NO:71. The AT domain contains the active site motif GxS*xG (SEQ ID NO:50), which is characteristic of acyltransferase (AT) proteins. The active site serine residue corresponds to S1121 of SEQ ID NO:71.

The fourth domain of Thraustochytrium ATCC PTA-10212 Pfa2 is the ER domain. The nucleotide sequence encoding the ER domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:108, corresponding to positions 4498-5700 of SEQ ID NO:70. The amino acid sequence encoding the ER domain of Thraustochytrium ATCC PTA-10212 Pfa2 is set forth herein as SEQ ID NO:109, corresponding to positions 1500-1900 of SEQ ID NO:71.

Table 11 shows the structural domains and active sites associated with Thraustochytrium ATCC PTA-10212 PFA3.

Table 11: Thraustochytrid ATCC PTA-10212 PFA3 domain analysis

The first and second domains of Thraustochytrium ATCC PTA-10212 Pfa3 are referred to herein as DH1 and DH2, respectively. The nucleotide sequence comprising the coding sequence for the DH1 domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO: 110, corresponding to positions 1-1350 of SEQ ID NO: 72. The amino acid sequence comprising the DH1 domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO: 111, corresponding to positions 1-450 of SEQ ID NO: 73. The nucleotide sequence comprising the coding sequence for the DH2 domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO: 112, corresponding to positions 1501-2700 of SEQ ID NO: 72. The amino acid sequence comprising the DH2 domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO:113 and corresponds to positions 501-900 of SEQ ID NO:73. The DH domain comprises the active site motif: FxxH*F (SEQ ID NO:53). The nucleotide sequence comprising the active site motif in DH1 corresponds to positions 934-936 of SEQ ID NO:72, while the nucleotide sequence comprising the active site motif in DH2 corresponds to positions 2401-2403 of SEQ ID NO:72. The active site H* in the motif FxxH*F is based on data from Leesong et al., Structure 4:253-64 (1996) and Kimber et al., J Biol Chem. 279:52593-602 (2004). The active site H* in DH1 corresponds to H312 of SEQ ID NO:73, and the active site H* in DH2 corresponds to H801 of SEQ ID NO:73.

The third domain of Thraustochytrium ATCC PTA-10212 Pfa3 is the ER domain. The nucleotide sequence encoding the ER domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO:114, corresponding to positions 2848-4200 of SEQ ID NO:72. The amino acid sequence encoding the ER domain of Thraustochytrium ATCC PTA-10212 Pfa3 is set forth herein as SEQ ID NO:115, corresponding to positions 950-1400 of SEQ ID NO:73.

Example 7

Inactivation of native PUFA synthase genes in Schizochytrium ATCC 20888 to generate PUFA auxotrophy and replacement of these inactivated genes with exogenously introduced homologous genes to restore PUFA synthesis have been previously demonstrated and described. See, for example, U.S. Patent No. 7,217,856, which is incorporated herein by reference in its entirety. The three PUFA synthase genes from Schizochytrium ATCC 20888 were previously designated orfA, orfB, and orfC, corresponding to the nomenclature used herein for PFA1, PFA2, and PFA3, respectively. Ibid.

After transformation with a vector containing the Zeocin ^™ resistance marker surrounded by orfA flanking region sequences, the native orfA gene of Schizochytrium sp. ATCC 20888 was replaced by homologous recombination. This resulted in a mutant lacking a functional orfA gene. This mutant is auxotrophic and requires PUFA supplementation for growth.

Schizochytrium sp. ATCC PTA-9695 PFA1 (SEQ ID NO: 1) was cloned into the expression vector pREZ37 to generate pREZ345. The expression vector contains approximately 2 kb of DNA flanking regions of the native orfA locus of Schizochytrium sp. ATCC 20888. pREZ345 containing PFA1 was used to transform a Schizochytrium sp. ATCC 20888 mutant lacking a functional orfA gene by electroporation with enzyme pretreatment (see below). Double crossover recombination occurred based on the flanking Zeocin ^™ resistance marker in the mutant and homologous regions flanking the PFA1 gene in pREZ345, resulting in the insertion of PFA1 into the native orfA locus. Recombination with Schizochytrium sp. ATCC PTA-9695 PFA1 (SEQ ID NO: 1) restored PUFA production in the Schizochytrium sp. ATCC 20888 mutant lacking orfA. Briefly, cells were cultured in M2B liquid medium (see below) at 30°C with shaking at 200 rpm for 3 days. Cells were harvested and fatty acids were converted to methyl esters using standard techniques. Fatty acid distribution as fatty acid methyl esters (FAMEs) was determined using gas chromatography with flame ionization detection (GC-FID). A native Schizochytrium sp. ATCC 20888 strain containing a functional orfA gene produced DHA and DPA in a ratio of 2.3:1. A recombinant strain in which the inactivated orfA gene was replaced with Schizochytrium sp. ATCC PTA-9695 PFA1 (SEQ ID NO: 1) also produced DHA and DPA n-6 in a ratio of 2.4:1. The recombinant strain had an EPA content of 2.7% fatty acid methyl esters (FAMEs), a DPAn-3 content of 0.7%, a DPA n-6 content of 8.8%, and a DHA content of 21.2%.

M2B medium—10 g/L glucose, 0.8 g/L (NH ₄ ) ₂ SO ₄ , 5 g/L Na ₂ SO ₄ , 2 g/L MgSO ₄ ·7H ₂ O, 0.5 g/L KH ₂ PO ₄ , 0.5 g/L KCl, 0.1 g/L CaCl ₂ ·2H ₂ O, 0.1 M MES (pH 6.0), 0.1% PB26 metals, and 0.1% PB26 vitamins (v/v). PB26 vitamins consist of 50 mg/mL vitamin B12, 100 μg/mL thiamine, and 100 μg/mL Ca-pantothenate. PB26 was adjusted to pH 4.5 and consisted of 3 g/L _FeSO₄ · _7H₂O , 1 g/ _L MnCl₂· _4H₂O , 800 mg/ _mL ZnSO₄· _7H₂O , 20 mg/mL _CoCl₂ ·6H₂O, _{10 mg} /mL _Na₂MoO₄ · _2H₂O , 600 mg/mL _CuSO₄ · _5H₂O , and 800 mg/mL NiSO₄· _6H₂O . The PB26 stock solution was filter sterilized separately and added to the broth after autoclaving. _Glucose , _KH₂PO₄ , and _CaCl₂ · _2H₂O were each autoclaved separately before mixing with _the other broth components to prevent salt precipitation and sugar caramelization. All media components were purchased from Sigma Chemical (St. Louis, MO).

Electroporation and Enzyme Pretreatment - Cells were grown in 50 mL of M50-20 medium (see U.S. Publication No. 2008/0022422) with shaking at 30°C for 2 days. Cells were diluted 1:100 with M2B medium and cultured overnight (16-24 hours) to mid-logarithmic phase (OD600 1.5-2.5). Cells were centrifuged at approximately 3000 x g for 5 minutes in a 50 mL conical tube. The supernatant was removed and the cells were resuspended in an appropriate volume of 1 M mannitol, pH 5.5, to a final concentration of 2 _OD600 units. Aliquot 5 mL of cells into a 25 mL shaker flask. Add 10 mM _CaCl₂ (1.0 M stock, filter sterilized) and 0.25 mg/mL Protease XIV (10 mg/mL stock, filter sterilized; Sigma-Aldrich, St. Louis, MO). Incubate the flask at 30°C with shaking at approximately 100 rpm for 4 hours. Observe microscopically for the extent of protoplast formation, ideally single cells. Centrifuge the cells at 2500 x g for 5 minutes in a round-bottom tube (i.e., a 14 mL Falcon ^™ tube; BD Biosciences, San Jose, CA). Remove the supernatant and gently resuspend the cells in 5 mL of ice-cold 10% glycerol. Centrifuge the cells again at approximately 2500 x g for 5 minutes in the round-bottom tube. Remove the supernatant and gently resuspend the cells in 500 μL of ice-cold 10% glycerol using a thick pipette tip. Aliquot 90 μL of cells into a pre-chilled electroporation cuvette (Gene Pulser cuvette - 0.1 cm gap or 0.2 cm gap, Bio-Rad, Hercules, CA) (Bio-Rad, Hercules, CA). Add 1-5 μg of DNA (10 μL volume or less) to the cuvette, gently mix with a pipette tip, and place on ice for 5 minutes. Electroporate the cells at 200 ohms (resistance), 25 μF (capacitance) and 250 V (0.1 cm gap) or 500 V (0.2 cm gap). Immediately add 0.5 mL of M50-20 medium to the cuvette. Then transfer the cells to a 25 mL shaking culture flask containing 4.5 mL of M50-20 medium and shake at approximately 100 rpm at 30°C for 2-3 hours. Centrifuge the cells in a round-bottom tube at approximately 2500 x g for 5 minutes. Remove the supernatant and resuspend the cell pellet in 0.5 mL of M50-20 medium. Plate the cells onto an appropriate number (2-5) of M2B plates containing the appropriate selection pressure and incubate at 30°C.

A Schizochytrium sp. ATCC 20888 mutant lacking a functional orfA was also transformed with pREZ345 containing PFAl to allow random integration of PFAl into the mutant and restore PUFA production.

Example 8

Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 68) (DNA2.0) was resynthesized and codon-optimized for expression in Schizochytrium (SEQ ID NO: 120), then cloned into the expression vector pLR95. Codon optimization was performed using the Schizochytrium codon usage table ( FIG. 22 ). The expression vector contained approximately 2 kb of DNA flanking the native orfA locus of Schizochytrium ATCC 20888.

The Schizochytrium ATCC 20888 mutant lacking a functional orfA gene from Example 7 was transformed with pLR95 containing a codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) by electroporation and enzyme pretreatment (see Example 7). Double crossover recombination occurred based on the flanking Zeocin ^™ resistance marker in the mutant and the homologous regions flanking the PFA1 gene in pLR95, resulting in insertion of the codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 into the native orfA locus. Recombination with the codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) restored PUFA production in the Schizochytrium ATCC 20888 mutant lacking orfA. Cell culture and FAME analysis were performed as in Example 7. A native Schizochytrium ATCC 20888 strain containing a functional orfA gene produced DHA and DPA in a ratio of 25:1. A recombinant strain in which the inactivated orfA gene was replaced with the codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) produced DHA and DPA in a ratio of 5.4:1, further demonstrating that the PUFA profile in Schizochytrium can be altered by the nucleic acid molecules described herein. The recombinant strain had an EPA content of 4.4% FAME, a DPA n-3 content of 2.3%, a DPA n-6 content of 4.9%, and a DHA content of 24.0%.

A Schizochytrium sp. ATCC 20888 mutant lacking a functional orfA was also transformed with pLR95 containing PFAl, resulting in random integration of PFAl into the mutant and restoration of PUFA production.

Example 9

After transformation with a vector containing the Zeocin ^™ resistance marker surrounded by orfB flanking region sequences via electroporation and enzyme pretreatment (see Example 7), the native orfB gene of Schizochytrium sp. ATCC 20888 was replaced by homologous recombination. This generated a mutant lacking a functional orfB gene. This mutant is auxotrophic and requires PUFA supplementation for growth.

Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3) was cloned into the expression vector pDS04 to generate pREZ331. The expression vector contains approximately 2 kb of DNA flanking regions of the native orfB locus of Schizochytrium ATCC 20888.

Schizochytrium ATCC 20888, which lacks a functional orfB, was transformed with pREZ331 containing PFA2. Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3) restored PUFA production based on random integration in the mutant.

Cell culture and FAME analysis were performed as in Example 7.

The native Schizochytrium ATCC 20888 strain, containing a functional orfB gene, produced DHA and DPA n-6 at a ratio of 2.3:1. A recombinant strain in which the inactivated orfB gene was replaced with Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3) produced DHA and DPA n-6 at a ratio of 3.5:1. The recombinant strain had an EPA content of 0.8% FAME, a DPA n-3 content of 0.1%, a DPA n-6 content of 7.1%, and a DHA content of 25.1%.

A Schizochytrium ATCC 20888 mutant lacking a functional orfB was also transformed with pREZ331 containing PFA2, thereby inserting PFA2 into the native orfB locus and restoring PUFA production.

Example 10

Thraustochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 70) (DNA2.0) was resynthesized and codon-optimized for expression in Schizochytrium (SEQ ID NO: 121), then cloned into the expression vector pLR85. Codon optimization was performed using the Schizochytrium codon usage table ( FIG. 22 ). The expression vector contained approximately 2 kb of DNA flanking regions of the native orfB locus of Schizochytrium ATCC 20888.

Orf gene replacement was also investigated in a daughter strain of Schizochytrium ATCC 20888 with enhanced DHA production. After transformation with a vector containing the Zeocin ^™ resistance marker surrounded by orfB flanking regions via electroporation and enzymatic pretreatment (see Example 7), the daughter strain's native orfB gene was replaced by homologous recombination. This generated a mutant lacking a functional orfB gene. This mutant is auxotrophic and requires PUFA supplementation for growth. The mutant was transformed with pLR85 containing the codon-optimized Thraustochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 121) via electroporation and enzymatic pretreatment (see Example 8). Double crossover recombination occurred based on the homologous regions flanking the Zeocin ^™ resistance marker in the mutant and the PFA2 gene in pLR85, resulting in insertion of the codon-optimized Thraustochytrium ATCC PTA-10212 PFA2 into the native orfB locus. Recombination with codon-optimized Thraustochytrium sp. ATCC PTA-10212 PFA2 (SEQ ID NO: 121) restored PUFA production in the substrain mutant lacking orfB. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 1.0% FAME, a DPA n-3 content of 0.3%, a DPA n-6 content of 7.0%, and a DHA content of 31.0%.

In the experiments performed, the Schizochytrium sp. ATCC 20888 mutant lacking a functional orfB gene from Example 9 was transformed with pLR85 containing a codon-optimized Thraustochytrium sp. ATCC PTA-10212 PFA2 (SEQ ID NO: 121) by electroporation and enzyme pretreatment (see Example 8). Double crossover recombination occurred based on the flanking Zeocin ^™ resistance marker in the mutant and the homologous regions flanking the PFA2 gene in pLR85, resulting in insertion of the codon-optimized Thraustochytrium sp. ATCC PTA-10212 PFA2 into the native orfB locus. Recombination with the codon-optimized Thraustochytrium sp. ATCC PTA-10212 PFA2 (SEQ ID NO: 121) restored PUFA production in the Schizochytrium sp. ATCC 20888 mutant lacking orfB.

Schizochytrium ATCC 20888 and a substrain mutant lacking a functional orfB were also transformed with pLR85 containing PFA2 to allow random integration of PFA2 into the mutants and restore PUFA production in each mutant.

Example 11

A plasmid containing a paromomycin resistance marker cassette functional in Schizochytrium was developed for Schizochytrium ATCC 20888 by replacing the bleomycin/zeocin ^™ resistance gene (ble) coding region in pMON50000/pTUBZEO11-2 with the coding region for neomycin phosphotransferase II (npt) derived from the bacterial transposon Tn5 (U.S. Patent 7,001,772 B2). In pMON50000, the ble resistance gene is driven by the Schizochytrium α-tubulin promoter and is followed by the SV40 transcriptional terminator. The ble region in pMON50000 contains an NcoI restriction site at the ATG start codon and a PmlI restriction site immediately following the TGA termination signal. The npt coding region from pCaMVnpt was amplified using PCR (Shimizu et al., Plant J. 26(4):375 (2001)) so that the product contained a BspHI restriction site (underlined below, primer CAX055) at the start ATG (bold) and a PmlI restriction site (underlined below, primer CAX056) immediately following the stop signal (bold - reverse complement):

CAX055 (forward): GTTGAACAAGATGGATTGCAC (SEQ ID NO: 66)

CAX056 (reverse): C CACGTG GAAGAACTCGTCAAGAA (SEQ ID NO: 67).

The product was cloned into pCR4-TOPO (Invitrogen) using the TaqMaster polymerase kit (5 Prime), and the resulting plasmid was used to transform Escherichia coli (E. coli) TOP10 (Invitrogen). DNA sequence analysis using vector primers identified multiple clones containing the desired 805 bp structure (i.e., sequences matching the source template plus the engineered restriction sites). The modified npt coding region was isolated by digestion with the BspHI and PmlI restriction enzymes, and the purified DNA fragment was ligated with the pMON50000 vector fragment generated by digestion with the NcoI and PmlI enzymes. The restriction enzymes BspHI and NcoI leave compatible overlapping ends, while PmlI leaves blunt ends. The resulting plasmid, pTS-NPT, contains the npt neomycin/paromomycin resistance gene and the same ble gene architecture as the original pMON50000.

The functionality of the new paromomycin resistance cassette in pTS-NPT was evaluated using Schizochytrium particle bombardment (U.S. Patent No. 7,001,772 B2). Selection for paromomycin (APR) resistance was performed on agar plates containing 50 μg/mL paromomycin sulfate (Sigma). The frequency of paromomycin-resistant Schizochytrium transformants was found to be similar to that of the Zeocin ^™ -resistant strain from pMON50000. The "α-tubulin promoter/npt/SV40 terminator" cassette can be released from pTS-NPT using a variety of restriction enzymes for subsequent use in other development applications.

Example 12

After transformation with a vector containing the Zeocin ^™ resistance marker surrounded by sequences from the orfC flanking regions, the native orfC gene of Schizochytrium sp. ATCC 20888 was replaced by homologous recombination. This resulted in a mutant lacking a functional orfC gene. This mutant is auxotrophic and requires PUFA supplementation for growth.

Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) was cloned into the expression vector pREZ22 to generate pREZ324. The expression vector contains approximately 2 kb of DNA flanking the native orfC locus of Schizochytrium ATCC 20888.

Schizochytrium ATCC 20888, lacking a functional orfC gene, was transformed with pREZ324 containing Schizochytrium ATCC PTA-9695 PFA3. Double crossover recombination occurred based on the flanking paromomycin resistance marker in the mutant and the homologous regions flanking the Schizochytrium ATCC PTA-9695 PFA3 gene in pREZ324, resulting in insertion of Schizochytrium ATCC PTA-9695 PFA3 into the native orfC locus. Homologous recombination with Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) restored PUFA production in the Schizochytrium ATCC 20888 mutant lacking orfC. Cell culture and FAME analysis were performed as in Example 7. The native Schizochytrium ATCC 20888 strain containing a functional orfC gene produced DHA and DPA n-6 in a ratio of 2.3:1. A recombinant strain in which the inactivated orfC gene was replaced with PFA3 (SEQ ID NO: 5) from Schizochytrium ATCC PTA-9695 produced DHA and DPA n-6 in a ratio of 14:9, further demonstrating that the PUFA profile of Schizochytrium can be altered by the nucleic acid molecules described herein. The recombinant strain had an EPA content of 1.2% FAME, a DPA n-3 content of 0.2%, a DPA n-6 content of 2.9%, and a DHA content of 43.4%.

A Schizochytrium sp. ATCC 20888 mutant lacking a functional orfC gene was also transformed with pREZ324 containing PFA3, thereby randomly integrating PFA3 into the mutant and restoring PUFA production. The recombinant strain had an EPA content of 1.2% FAME, a DPA n-3 content of 0.2%, a DPA n-6 content of 2.5%, and a DHA content of 39.1%.

After transformation with a vector containing a paromomycin resistance marker surrounded by orfC flanking region sequences, the native orfC gene of the daughter strain discussed in Example 10 was replaced by homologous recombination. This generated a mutant lacking a functional orfC gene. This mutant is auxotrophic and requires PUFA supplementation for growth. The mutant lacking a functional orfC gene was transformed with pREZ324. Double crossover recombination occurred, resulting in the insertion of Schizochytrium sp. ATCC PTA-9695 PFA3 into the mutant's native orfC locus. Homologous recombination with Schizochytrium sp. ATCC PTA-9695 PFA3 (SEQ ID NO: 5) restored PUFA production in the daughter strain lacking orfC. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 1.2% FAME, a DPA n-3 content of 0.3%, a DPA n-6 content of 2.8%, and a DHA content of 43.1%.

A daughter strain mutant lacking a functional orfB was also transformed with pREZ324 containing PFA3, resulting in random integration of PFA3 into the mutant and restoration of PUFA production.

Example 13

Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 72) (DNA2.0) was resynthesized and codon-optimized for expression in Schizochytrium (SEQ ID NO: 122) and cloned into the expression vector pREZ22 to generate pREZ337. Codon optimization was performed using the Schizochytrium codon usage table ( FIG. 22 ). The expression vector contained approximately 2 kb of DNA flanking regions of the native orfC locus of Schizochytrium ATCC 20888.

The substrain mutant lacking a functional orfC gene described in Example 12 (see Example 8) was transformed using pREZ337 containing a codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) by electroporation and enzyme pretreatment. Double crossover recombination occurred based on the flanking Zeocin ^™ resistance marker in the mutant and the homologous regions flanking the PFA3 gene in pREZ337, resulting in insertion of the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) into the native orfC locus. Recombination with the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) restored PUFA production in the substrain mutant lacking orfC. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 1.3% FAME, a DPA n-3 content of 0.4%, a DPA n-6 content of 2.7% and a DHA content of 50.2%.

In the experiments performed, the Schizochytrium ATCC 20888 mutant lacking a functional orfC gene from Example 12 was transformed with pREZ337 containing a codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) by electroporation and enzyme pretreatment (see Example 8). Double crossover recombination occurred based on the flanking Zeocin ^™ resistance marker in the mutant and the homologous regions flanking the PFA3 gene in pREZ337, resulting in insertion of the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) into the native orfC locus. Recombination with the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) restored PUFA production in the Schizochytrium ATCC 20888 mutant lacking orfC.

Schizochytrium ATCC 20888 and substrain mutants lacking functional orfC were also transformed with pREZ337 containing PFA3 to allow random integration of PFA3 into the mutants and restore PUFA production in each mutant.

Example 14

After transforming Schizochytrium sp. ATCC 20888 with a vector containing the Zeocin ^™ or Paromomycin resistance marker surrounded by sequences of the appropriate orf flanking regions, any two or all three of orfA, orfB, and orfC are replaced by homologous recombination. This results in a mutant strain lacking any two or all three functional genes among orfA, orfB, and orfC. This mutant strain is auxotrophic and requires PUFA supplementation for growth.

A Schizochytrium sp. ATCC 20888 mutant lacking a functional orf gene was transformed with one or more expression vectors containing the corresponding PFA gene (one or more of SED ID NOs: 1, 3, 5, 120, 121, or 122). Double crossover recombination occurred between the flanking Zeocin ^™ or Paromomycin resistance marker in the mutant and the homologous regions flanking the PFA gene in each vector, resulting in insertion of the PFA gene into the native orf locus. Random integration of these expression vectors also allowed selection of transformants based solely on restoration of PUFA production. Homologous recombination with the PFA gene restored PUFA production in the mutant, thereby restoring or altering the native PUFA profile based on the combination of PFA genes inserted into the mutant.

In the experiments performed, the Schizochytrium ATCC 20888 strain lacking a functional orfC gene and containing randomly integrated Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) from Example 12 was used to replace orfA and orfB. Following transformation with a vector containing the Zeocin ^™ resistance marker surrounded by sequences flanking the orfA and orfB regions, the strain's native orfA and orfB genes were replaced by homologous recombination. The resulting strain lacked functional orfA, orfB, and contained randomly integrated Schizochytrium ATCC PTA-9695 PFA3. The strain was transformed with pREZ345, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA1 (SEQ ID NO: 1), and pREZ331, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3), to randomly integrate PFA1 and PFA2. The resulting recombinant strain lacking functional orfA, orfB, and orfC contained randomly integrated Schizochytrium sp. ATCC PTA-9695 PFA1, PFA2, and PFA3. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 6.6% FAME, a DPA n-3 content of 0.8%, a DPA n-6 content of 1.6%, and a DHA content of 20.9%.

In another experiment, a substrain of Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) from Example 12, lacking a functional orfC gene and inserted into the native orfC locus, was used to replace the orfA and orfB genes. Following transformation with a vector containing a paromomycin resistance marker surrounded by orfA and orfB flanking region sequences, the strain's native orfA and orfB genes were replaced by homologous recombination. The resulting strain lacked functional orfA, orfB, and contained Schizochytrium ATCC PTA-9695 PFA3 inserted into the native orfC locus. The strain was transformed with pREZ345, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA1 (SEQ ID NO: 1), and pREZ331, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3). Double crossover recombination occurred, resulting in the insertion of Schizochytrium ATCC PTA-9695 PFA1 into the strain's native orfA locus, and the insertion of Schizochytrium ATCC PTA-9695 PFA2 into the strain's native orfB locus. The resulting recombinant strain lacked functional orfA, orfB, and orf, and contained Schizochytrium ATCC PTA-9695 PFA1, PFA2, and PFA3, each inserted into the orfA, orfB, and orfC loci, respectively. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 7.3% FAME, a DPA n-3 content of 0.4%, a DPA n-6 content of 1.5%, and a DHA content of 23.9%.

In another experiment, a daughter strain from Example 12 lacking a functional orfC gene and containing randomly integrated Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) was used to replace the orfA and orfB genes. Following transformation with a vector containing the Zeocin ^™ resistance marker surrounded by orfA and orfB flanking region sequences, the strain's native orfA and orfB genes were replaced by homologous recombination. The resulting strain lacked functional orfA, orfB, and contained randomly integrated Schizochytrium ATCC PTA-9695 PFA3. The strain was transformed with pREZ345, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA1 (SEQ ID NO: 1), and pREZ331, containing codon-optimized Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3), to randomly integrate PFA1 and PFA2. The resulting recombinant strain lacking functional orfA, orfB, and orfC contained randomly integrated Schizochytrium sp. ATCC PTA-9695 PFA1, PFA2, and PFA3. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 6.2% FAME, a DPA n-3 content of 1.3%, a DPA n-6 content of 0.9%, and a DHA content of 16.6%.

In another experiment, a daughter strain of Example 13 lacking a functional orfC gene and containing Schizochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) inserted into the native orfC locus was used to replace the orfA and orfB genes. Following transformation with a vector containing a paromomycin resistance marker surrounded by orfA and orfB flanking region sequences, the strain's native orfA and orfB genes were replaced by homologous recombination. The resulting strain lacked functional orfA, orfB, and contained Schizochytrium ATCC PTA-10212 PFA3 inserted into the native orfC locus. The strain was transformed with pLR95 containing codon-optimized Schizochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) and pLR85 containing codon-optimized Schizochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 121). Double crossover recombination occurred, resulting in the insertion of Schizochytrium ATCC PTA-10212 PFA1 into the strain's native orfA locus, and the insertion of Schizochytrium ATCC PTA-10212 PFA2 into the strain's native orfB locus. The resulting recombinant strain lacked functional orfA, orfB, and orf, and contained Schizochytrium ATCC PTA-10212 PFA1, PFA2, and PFA3, each inserted into the orfA, orfB, and orfC loci, respectively. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 5.2% FAME, a DPA n-3 content of 0.6%, a DPA n-6 content of 2.1%, and a DHA content of 47.1%.

In another experiment, a daughter strain from Example 13 lacking a functional orfC gene and containing randomly integrated Schizochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) was used to replace the orfA and orfB genes. Following transformation with a vector containing the Zeocin ^™ resistance marker surrounded by orfA and orfB flanking region sequences, the strain's native orfA and orfB genes were replaced by homologous recombination. The resulting strain lacked functional orfA, orfB, and contained randomly integrated Schizochytrium ATCC PTA-10212 PFA3. The strain was transformed with pLR95 containing codon-optimized Schizochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) and pLR85 containing codon-optimized Schizochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 121) to randomly integrate PFA1 and PFA2. The resulting recombinant strain lacking functional orfA, orfB, and orfC contained randomly integrated Schizochytrium sp. ATCC PTA-10212 PFA1, PFA2, and PFA3. Cell culture and FAME analysis were performed as in Example 7. The recombinant strain had an EPA content of 1.8% FAME, a DPA n-3 content of 1.8%, a DPA n-6 content of 2.3%, and a DHA content of 34.1%.

Example 15

The orfA, orfB, and orfC genes from Schizochytrium ATCC 20888 were cloned into a series of dual expression vectors (Novagen). The dual expression vectors are a set of compatible plasmids in which multiple target genes are cloned and co-expressed from the T7 inducible promoter of E. coli. The dual plasmid pREZ91 contains Schizochytrium ATCC 20888 orfA in pETDuet-1; the dual plasmid pREZ96 contains Schizochytrium ATCC 20888 orfB in pCDFDuet-1; and the dual plasmid pREZ101 contains Schizochytrium ATCC 20888 orfC in pCOLADuet-1. The binary plasmids pREZ91, pREZ96, and pREZ101, as well as the plasmid pJK737 containing the desired accessory gene HetI (as described in U.S. Patent 7,217,856, incorporated by reference in its entirety), were transformed into E. coli strain BLR(DE3) containing the inducible T7 RNA polymerase gene. DHA and DPA n-6 were produced following cell growth and the addition of IPTG according to the manufacturer's instructions (Novagen). Briefly, 1 mM IPTG was added for induction when the cells reached an optical density at 600 nm of approximately 0.5. The cells were grown in Luria broth at 30°C for 12 hours and then harvested. The fatty acids were converted to methyl esters using standard techniques. The fatty acid profile was determined as fatty acid methyl esters (FAMEs) using gas chromatography equipped with flame ionization detection (GC-FID).

The Schizochytrium ATCC PTA-9695 PFA1 (SEQ ID NO: 1) gene was cloned into the expression vector pETDuet-1 to generate pREZ346. The binary plasmids pREZ346 (containing Schizochytrium ATCC PTA-9695 PFA1), pREZ96 (containing orfB), and pREZ101 (containing orfC) were transformed with pJK737 (containing HetI) into E. coli strain BLR(DE3). The Schizochytrium ATCC PTA-9695 PFA1 gene was co-expressed with the Schizochytrium ATCC 20888 orfB and orfC genes. The Schizochytrium ATCC PTA-9695 PFA1 and the Schizochytrium ATCC 20888 orfB and orfC genes supported DHA production in E. coli when expressed under inducing conditions. The DHA content of the transformed E. coli was 2.8% FAME, the DPA n-6 content was 1.1%, the DPA n-3 content was 0.6% and the EPA content was 3.7%.

Example 16

The codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 (SEQ ID NO: 120) gene was cloned into the expression vector pETDuet-1 to generate pLR100. The binary plasmids pLR100 (containing the codon-optimized Thraustochytrium ATCC PTA-10212 PFA1), pREZ96 (containing orfB from Schizochytrium ATCC 20888), pREZ101 (containing orfC from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. The Thraustochytrium ATCC PTA-10212 PFA1 was co-expressed with the Schizochytrium ATCC 20888 orfB and orfC genes. The expression of orfB and orfC of Thraustochytrium ATCC PTA-10212 PFA1 and Schizochytrium ATCC 20888 under inducible conditions supported the production of DHA and EPA in E. coli.

Example 17

The Schizochytrium ATCC PTA-9695 PFA3 (SEQ ID NO: 5) gene was cloned into the expression vector pCOLADuet-1 to generate pREZ326. The binary plasmids pREZ326 (containing Schizochytrium ATCC PTA-9695 PFA3), pREZ91 (containing orfA from Schizochytrium ATCC PTA-20888), pREZ96 (containing orfB from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of Schizochytrium ATCC PTA-9695 PFA3 and Schizochytrium ATCC 20888 orfA and orfB under inducing conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The DHA content of the transformed E. coli was 0.3% FAME.

Example 18

The codon-optimized Thraustochytrium ATCC PTA-10212 PFA3 (SEQ ID NO: 122) gene was cloned into the expression vector pCOLADuet-1 to generate pREZ348. The binary plasmids pREZ348 (containing the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3), pREZ91 (containing orfA from Schizochytrium ATCC 20888), pREZ96 (containing orfB from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of the Thraustochytrium ATCC PTA-10212 PFA3 and Schizochytrium ATCC 20888 orfA and orfB under inducible conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as described in Example 15. The DHA content of the transformed E. coli was 2.9% FAME, and the DPA n-6 content was 0.4%.

Example 19

The Schizochytrium ATCC PTA-9695 PFA2 (SEQ ID NO: 3) gene was cloned into the expression vector pCDFDuet-1 to generate pREZ330. The binary plasmids pREZ330 (containing Schizochytrium ATCC PTA-9695 PFA2), pREZ326 (containing Schizochytrium ATCC PTA-9695 PFA3), pREZ91 (containing orfA from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 9. Expression of Schizochytrium ATCC PTA-9695 PFA2 and PFA3, as well as Schizochytrium ATCC 20888 orfA, under inducible conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The transformed E. coli had a DHA content of 0.8% FAME and a DPA n-6 content of 0.2%.

Example 20

The codon-optimized Thraustochytrium ATCC PTA-10212 PFA2 (SEQ ID NO: 121) gene was cloned into the expression vector pCDFDuet-1 to generate pLR87. The binary plasmids pLR87 (containing the codon-optimized Schizochytrium ATCC PTA-10212 PFA2), pREZ348 (containing the codon-optimized Thraustochytrium ATCC PTA-10212 PFA3), pREZ91 (containing orfA from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. Expression of Thraustochytrium ATCC PTA-10212 PFA2 and PFA3, as well as Schizochytrium ATCC 20888 orfA, under inducing conditions supported DHA and low-level EPA production in E. coli. Cell culture and FAME analysis were performed as described in Example 15. The DHA content of the transformed E. coli was 4.4% FAME, the DPA n-6 content was 1.1%, and the EPA content was 0.1%.

Example 21

The binary plasmids pREZ346 (containing Schizochytrium ATCC PTA-9695 PFA1), pREZ330 (containing Schizochytrium ATCC PTA-9695 PFA2), pREZ326 (containing Schizochytrium ATCC PTA-9695 PFA3), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. Expression of Schizochytrium ATCC PTA-9695 PFA1, PFA2, and PFA3 under inducing conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The DHA content of the transformed E. coli was 0.3% FAME and the EPA content was 0.3%.

Example 22

The binary plasmids pLR100 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA1), pLR87 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA2), pREZ348 (containing PFA3 of Schizochytrium ATCC PTA-10212), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. Expression of Thraustochytrium ATCC PTA-10212 PFA1, PFA2, and PFA3 under inducing conditions supported the production of DHA and EPA in E. coli.

Example 23

The binary plasmids pREZ330 (containing Schizochytrium ATCC PTA-9695 PFA2), pREZ91 (containing Schizochytrium ATCC 20888 orfA), pREZ101 (containing Schizochytrium ATCC 20888 orfC), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of Schizochytrium ATCC PTA-9695 PFA2 and Schizochytrium ATCC 20888 orfA and orfC under inducible conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The DHA content of the transformed E. coli was 0.6% FAME and the DPA n-6 content was 0.3%.

Example 24

The binary plasmids pLR187 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA2), pREZ91 (containing orfA from Schizochytrium ATCC 20888), pREZ101 (containing orfC from Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of codon-optimized Thraustochytrium ATCC PTA-10212 PFA2 and Schizochytrium ATCC 20888 orfA and orfC under inducible conditions supported DHA and low-level EPA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The transformed E. coli had a DHA content of 1.7% FAME, a DPA n-6 content of 0.9%, and an EPA content of 0.1%.

Example 25

The binary plasmids pREZ346 (containing Schizochytrium ATCC PTA-9695 PFA1), pREZ330 (containing Schizochytrium ATCC PTA-9695 PFA2), pREZ101 (containing Schizochytrium ATCC 20888 orfC), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of PFA1 and PFA2, as well as Schizochytrium ATCC 20888 orfC, under inducible conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The transformed E. coli had a DHA content of 0.3% FAME, a DPA n-6 content of 0.1%, and an EPA content of 0.5%.

Example 26

The binary plasmids pLR100 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA1), pLR87 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA2), pREZ101 (containing orfC of Schizochytrium ATCC 20888), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. Expression of codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 and PFA2 and Schizochytrium ATCC 20888 orfC under inducing conditions supported the production of DHA and EPA in E. coli.

Example 27

The binary plasmids pREZ346 (containing Schizochytrium ATCC PTA-9695 PFA1), pREZ96 (containing Schizochytrium ATCC 20888 orfB), pREZ326 (containing Schizochytrium ATCC PTA-9695 PFA3), and pJK737 (containing HetI) were transformed into E. coli strain BLR(DE3). See Example 15. Expression of Schizochytrium ATCC PTA-9695 PFA1 and PFA3, as well as Schizochytrium ATCC 20888 orfB, under inducible conditions supported DHA production in E. coli. Cell culture and FAME analysis were performed as in Example 15. The DHA content of the transformed E. coli was 0.1% FAME and the EPA content was 0.1%.

Example 28

The binary plasmids pLR100 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA1), pREZ96 (containing orfB of Schizochytrium ATCC 20888), pREZ348 (containing codon-optimized Thraustochytrium ATCC PTA-10212 PFA3), and pJK737 (containing HetI) were transformed together into E. coli strain BLR(DE3). See Example 15. Expression of codon-optimized Thraustochytrium ATCC PTA-10212 PFA1 and PFA3 and Schizochytrium ATCC 20888 orfB under inducing conditions supported the production of DHA and EPA in E. coli.

Example 29

The Pfa1p, Pfa2p, and Pfa3p PUFA synthase activities were individually knocked out in Schizochytrium ATCC PTA-9695 and Thraustochytrium ATCC PTA-10212 by standard procedures. See, eg, US Patent No. 7,217,856, which is incorporated herein by reference in its entirety.

Zeocin ^™ , hygromycin, blasticidin, or another suitable resistance marker is inserted into the restriction site of the PFA1 gene (SEQ ID NO: 1 or SEQ ID NO: 68) contained in the plasmid. After insertion of the resistance marker, the plasmid is introduced into Schizochytrium sp. ATCC PTA-9695 or Thraustochytrium sp. ATCC PTA-10212, respectively, by particle bombardment, electroporation, or another suitable transformation method. Homologous recombination occurs, generating mutants in which the native PFA1 gene is replaced or disrupted with Zeocin ^™ , hygromycin, blasticidin, or another suitable resistance marker. Transformants are selected on plates containing Zeocin ^™ , hygromycin, blasticidin, or another suitable selective agent and supplemented with PUFAs. Clones are further tested for their growth ability without PUFA supplementation. Genomic DNA is extracted from clones that are resistant to the selective agent and unable to grow in the absence of PUFAs. PCR and Southern blot analysis of this DNA confirms the deletion or disruption of the PFA1 gene.

A similar procedure was used to knock out PFA2. The resulting knockout mutant, which required PUFA supplementation, was found to lack full-length PFA2.

A similar procedure was used to knock out PFA3. The resulting knockout mutant, which required PUFA supplementation, was found to lack full-length PFA3.

All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims (29)

1. An isolated nucleic acid molecule having a polynucleotide sequence that is 100% identical to SEQ ID NO: 37 or SEQ ID NO: 5, wherein the nucleic acid molecule encodes a polypeptide containing polyunsaturated fatty acid (PUFA) synthase activity. 2. The isolated nucleic acid molecule according to claim 1, wherein the polynucleotide sequence of the nucleic acid molecule is SEQ ID NO: 5. 3. An isolated nucleic acid molecule whose polynucleotide sequence encodes a polypeptide that is 100% identical to SEQ ID NO: 38 or SEQ ID NO: 6, wherein the polypeptide contains polyunsaturated fatty acid (PUFA) synthase activity. 4. The isolated nucleic acid molecule according to claim 3, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 6. 5. An isolated nucleic acid molecule whose polynucleotide sequence is completely complementary to the polynucleotide sequence of any one of claims 1-4. 6. A recombinant nucleic acid molecule comprising the nucleic acid molecule or combination thereof as described in any one of claims 1-4 and a transcription control sequence. 7. The recombinant nucleic acid molecule according to claim 6, wherein the recombinant nucleic acid molecule is a recombinant vector. 8. A host cell expressing a nucleic acid molecule as described in any one of claims 1-4, a recombinant nucleic acid molecule as described in claim 6 or 7, or a combination thereof, wherein the host cell is selected from microbial cells and animal cells. 9. The host cell according to claim 8, wherein the host cell is an animal cell. 10. The host cell according to claim 8, wherein the host cell is a microbial cell. 11. The host cell as claimed in claim 10, wherein the microbial cell is a bacterium. 12. The host cell as claimed in claim 10, wherein the microbial cell is *Cyclochytrium*. 13. The host cell according to claim 12, wherein the schistocytic fungus is a member of the genus Schistocyticum or the genus Schistocyticum. 14. A method for generating at least one PUFA, comprising: Under conditions that effectively produce PUFA, the PUFA synthase gene is expressed in the host cell. The host cell comprises the isolated nucleic acid molecule of any one of claims 1-4, the recombinant nucleic acid molecule of claim 6 or 7, or a combination thereof, and At least one PUFA is produced. 15. The method of claim 14, wherein the host cell is selected from plant cells, isolated animal cells, and microbial cells. 16. The method of claim 14, wherein the at least one PUFA contains docosahexaenoic acid (DHA). 17. A method for producing DHA-rich lipids, comprising: Under conditions conducive to lipid production, the PUFA synthase gene is expressed in the host cells of any one of claims 8-13, and It produces lipids rich in DHA. 18. A method for producing a recombinant vector, comprising inserting the isolated nucleic acid molecule of any one of claims 1-4 into the vector. 19. A method for generating recombinant host cells, comprising introducing the recombinant vector of claim 18 into a host cell. 20. The method of claim 19, wherein the host cell is selected from plant cells, isolated animal cells, and microbial cells. 21. An isolated polypeptide, wherein the polypeptide is encoded by the polynucleotide sequence of any one of claims 1-4. 22. An isolated polypeptide having an amino acid sequence that is 100% identical to SEQ ID NO: 38, wherein the polypeptide contains polyunsaturated fatty acid (PUFA) synthase activity. 23. An isolated polypeptide having an amino acid sequence that is 100% identical to SEQ ID NO: 6, wherein the polypeptide contains polyunsaturated fatty acid (PUFA) synthase activity. 24. The isolated polypeptide according to any one of claims 22-23, wherein the polypeptide is a fusion polypeptide. 25. A composition comprising the polypeptide of any one of claims 22-23 and a biologically acceptable carrier. 26. A method for increasing DHA production in an organism possessing PUFA synthase activity, comprising: Under conditions that effectively produce DHA, the isolated nucleic acid molecule of any one of claims 1-4, the recombinant nucleic acid molecule of claim 6 or 7, or a combination thereof, is expressed in the organism. The PUFA synthase activity described herein replaces inactivated or missing activity in the organism, introduces new activity, or enhances existing activity, and The DHA production in the organism increases. 27. A method for isolating lipids from host cells, comprising: (a) Under conditions conducive to lipid production, the PUFA synthase gene is expressed in the host cell, wherein the polyketide synthase system in the host cell comprises the isolated nucleic acid molecule of any one of claims 1-4, the recombinant nucleic acid molecule of claim 6 or 7, or a combination thereof, and (b) Isolate lipids from the host cells. 28. The method of claim 27, wherein the host cell is selected from plant cells, isolated animal cells, and microbial cells. 29. The method of claim 27, wherein the lipid comprises DHA.