Elongase genes and uses thereof

Abstract

The subject invention relates to the identification of several genes involved in the elongation of polyunsaturated acids (i.e., “elongases”) and to uses thereof. At least two of these genes are also involved in the elongation of monounsaturated fatty acids. In particular, elongase is utilized in the conversion of gamma linolenic acid (GLA) to dihomogamma linolenic acid (DGLA) and in the conversion of AA to adrenic acid (ADA), or eicosapentaenoic acid (EPA) to ω3-docosapentaenoic acid (DPA). DGLA may be utilized in the production of polyunsaturated fatty acids, such as arachidonic acid (AA), docosahexaenoic acid (DHA), EPA, adrenic acid, ω6-docosapentaenoic acid or ω3-docosapentaenoic acid which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

1 . An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence encoding a polypeptide having elongase activity, wherein the amino acid sequence of said polypeptide has at least 50% identity to an amino acid sequence comprising SEQ ID NO:121.  
     
     
         2 . An isolated nucleotide sequence or fragment thereof comprising or complementary to a nucleotide sequence having at least 50% nucleotide sequence identity to a nucleotide sequence comprising SEQ ID NO:119.  
     
     
         3 . The isolated nucleotide sequence of claims  1  or  2  wherein said sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate.  
     
     
         4 . The isolated nucleotide sequence of  claim 1  wherein said sequence is derived from an algae.  
     
     
         5 . The isolated nucleotide sequence of  claim 4  wherein said algae is Pavlova sp.  
     
     
         6 . A purified protein encoded by said nucleotide sequence of claims  1  or  2 .  
     
     
         7 . A purified polypeptide which elongates polyunsaturated fatty acids and has at least about 30% amino acid identity to the amino acid sequence of said purified protein of  claim 6 .  
     
     
         8 . A method of producing an elongase enzyme comprising the steps of: 
 a) isolating a nucleotide sequence comprising SEQ ID NO:119;    b) constructing a vector comprising: i) said isolated nucleotide sequence operably linked to ii) a promoter;    c) introducing said vector into a host cell under time and conditions sufficient for expression of said elongase enzyme.    
     
     
         9 . The method of  claim 8  wherein said host cell is selected from the group consisting of a eukaryotic cell or a prokaryotic cell.  
     
     
         10 . The method of  claim 9  wherein said prokaryotic cell is selected from the group consisting of  E. coli,  Cyanobacteria, and  B. subtilis.    
     
     
         11 . The method of  claim 9  wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.  
     
     
         12 . The method of  claim 11  wherein said fungal cell is selected from the group consisting of Saccharomyces spp., Candida spp.,  Lipomyces starkey , Yarrowia spp., Kluyveromyces spp., Hansenula spp., Aspergillus spp., Penicillium spp., Neurospora spp., Trichoderma spp. and Pichia spp.  
     
     
         13 . The method of  claim 12  wherein said fungal cell is a yeast cell selected from the group consisting of Saccharomyces spp., Candida spp., Hansenula spp. and Pichia spp.  
     
     
         14 . The method of  claim 13  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         15 . A vector comprising: a) a nucleotide sequence comprising SEQ ID NO:119 operably linked to b) a promoter.  
     
     
         16 . A host cell comprising said vector of  claim 15 .  
     
     
         17 . The host cell of  claim 16  wherein said host cell is selected from the group consisting of a eukaryotic cell or a prokaryotic cell.  
     
     
         18 . The host cell of  claim 17  wherein said prokaryotic cell is selected from the group consisting of  E. coli , Cyanobacteria, and  B. subtilis.    
     
     
         19 . The host cell of  claim 17  wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell.  
     
     
         20 . The host cell of  claim 19  wherein said fungal cell is selected from the group consisting of Saccharomyces spp., Candida spp.,  Lipomyces starkey , Yarrowia spp., Kluyveromyces spp., Hansenula spp., Aspergillus spp., Penicillium spp., Neurospora spp., Trichoderma spp. and Pichia spp.  
     
     
         21 . The host cell of  claim 20  wherein said fungal cell is a yeast cell selected from the group consisting of Saccharomyces spp., Candida spp., Hansenula spp. and Pichia spp.  
     
     
         22 . The host cell of  claim 21  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         23 . A plant cell, plant or plant tissue comprising said vector of  claim 15 , wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid by said plant cell, plant or plant tissue.  
     
     
         24 . The plant cell, plant or plant tissue of  claim 23  wherein said polyunsaturated fatty acid is selected from the group consisting of ADA and ω3-docosapentaenoic acid.  
     
     
         25 . One or more plant oils or acids expressed by said plant cell, plant or plant tissue of  claim 23 .  
     
     
         26 . A transgenic plant comprising said vector of  claim 15 , wherein expression of said nucleotide sequence of said vector results in production of a polyunsaturated fatty acid in seeds of said transgenic plant.  
     
     
         27 . A transgenic, non-human mammal whose genome comprises a DNA sequence encoding an elongase, operably linked to a promoter, wherein said DNA sequence comprises SEQ ID NO:119.  
     
     
         28 . A fluid produced by said transgenic, non-human mammal of  claim 27  wherein said fluid comprises a detectable level of at least one elongase or products thereof.  
     
     
         29 . A method for producing a polyunsaturated fatty acid comprising the steps of: 
 a) isolating a nucleotide sequence comprising SEQ ID NO:119;    b) constructing a vector comprising said isolated nucleotide sequence;    c) introducing said vector into a host cell under time and conditions sufficient for expression of an elongase enzyme encoded by said isolated nucleotide sequence; and    d) exposing said expressed elongase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.    
     
     
         30 . The method according to  claim 29 , wherein said substrate polyunsaturated fatty acid is selected from the group consisting of AA and EPA, and said product polyunsaturated fatty acid is selected from the group consisting of ADA and ω3-docosapentaenoic acid, respectively.  
     
     
         31 . The method according to  claim 29  further comprising the step of exposing said expressed elongase enzyme to at least one desaturase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid.  
     
     
         32 . The method according to  claim 31  wherein said product polyunsaturated fatty acid is selected from the group consisting of ADA and ω3-docosapentaenoic acid, said another polyunsaturated fatty acid is selected from the group consisting of ω6-docosapentaenoic acid, ω3-docosapentaenoic acid and docohexaenoic acid, and said at least one desaturase is Δ4-desaturase with respect to production of ω6-docosapentaenoic acid or docosahexaenoic acid, and Δ19-desaturase with respect to production of ω3-docosapentaenoic acid or docosahexaenoic acid.  
     
     
         33 . The method of  claim 32  further comprising the step of exposing said another polyunsaturated fatty acid to one or more enzymes selected from the group consisting of at least one elongase and at least one additional desaturase in order to convert said another polyunsaturated fatty acid to a final polyunsaturated fatty acid.  
     
     
         34 . The method of  claim 33  wherein said final polyunsaturated fatty acid is docosahexaenoic acid.  
     
     
         35 . A nutritional composition comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of  claim 29 , said another polyunsaturated fatty acid produced according to the method of  claim 31 , and said final polyunsaturated fatty acid produced according to the method of  claim 33 .  
     
     
         36 . The nutritional composition of  claim 35  wherein said product polyunsaturated fatty acid is selected from the group consisting of ADA and ω3-docosapentaenoic.  
     
     
         37 . The nutritional composition of  claim 35  wherein said another polyunsaturated fatty acid is selected from the group consisting ω6-docosapentaenoic acid, ω3-docosapentaenoic acid and docosahexaenoic acid.  
     
     
         38 . The nutritional composition of  claim 35  wherein said final polyunsaturated fatty acid is docosahexaenoic acid.  
     
     
         39 . The nutritional composition of  claim 35  wherein said nutritional composition is selected from the group consisting of an infant formula, a dietary supplement and a dietary substitute.  
     
     
         40 . A pharmaceutical composition comprising 1) at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of  claim 29 , said another polyunsaturated fatty acid produced according to the method of  claim 31 , and said final polyunsaturated fatty acid produced according to the method of  claim 33  and 2) a pharmaceutically acceptable carrier.  
     
     
         41 . An animal feed comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of  claim 29 , said another polyunsaturated fatty acid produced according to the method of  claim 31  and said final polyunsaturated fatty acid produced according to the method of  claim 33 .  
     
     
         42 . A method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to said patient said nutritional composition of  claim 35  in an amount sufficient to effect said prevention or treatment.