Processes for producing polyunsaturated fatty acids in transgenic organisms

Abstract

The present invention relates to polynucleotides from Ostreococcus lucimarinus which code for desaturases and elongases and which can be employed for the recombinant production of polyunsaturated fatty acids. The invention furthermore relates to vectors, host cells and transgenic nonhuman organisms which comprise the polynucleotides, and to the polypeptides encoded by the polynucleotides. Finally, the invention also relates to production processes for the polyunsaturated fatty acids and for oil, lipid and fatty acid compositions.

We claim: 
     
         1 . A process for the production of a substance which has the structure shown in the general formula I hereinbelow: 
       
         
           
           
               
               
           
         
         where the variables and substituents are as follows:
 R 1 =hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the formula II 
 
       
       
         
           
           
               
               
           
         
         
           R 2 =hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl, 
           R 3 =hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl, 
           or R 2  and R 3  independently of one another are a radical of the formula Ia: 
         
       
       
         
           
           
               
               
           
         
         
           in which 
           n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3; and wherein the process comprises the cultivation of:
 (i) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with  Ostreococcus lucimarinus -CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleic acid sequences of (a) or (b), and 
 wherein the polynucleotide is operatively linked to an expression control sequence; or 
 
 (ii) a transgenic, nonhuman organism comprising:
 I) an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with Ostreococcus lucimannus CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and 
 wherein the polynucleotide is operatively linked to an expression control sequence; 
 II) a vector comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with  Ostreococcus lucimarinus  CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and 
 
 wherein the polynucleotide is operatively linked to an expression control sequence, or
 III) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with  Ostreococcus lucimarinus  CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and 
 
 
           wherein the polynucleotide is operatively linked to an expression control sequence, 
         
         under conditions which permit the biosynthesis of the substance. 
       
     
     
         2 . A process for the production of a substance which has the structure shown in the general formula I hereinbelow: 
       
         
           
           
               
               
           
         
         where the variables and substitutents are as follows:
 R 1 =hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the formula II 
 
       
       
         
           
           
               
               
           
         
         
           R 2 =hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl, 
           R 3 =hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl, 
           or R 2  and R 3  independently of one another are a radical of the formula Ia: 
         
       
       
         
           
           
               
               
           
         
         
           n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3; and wherein the process comprises the cultivation of:
 (i) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as  Ostreococcus lucimarinus  CoA-dependent delta 6 desaturase, which nucleic acid sequence comprises
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleic acid sequences of (a) or (b) 
 
 
           and wherein the polynucleotide is operatively linked to an expression control sequence; or
 (ii) a transgenic, nonhuman organism comprising:
 I) an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as  Ostreococcus lucimarinus  CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and 
 wherein the polynucleotide is operatively linked to an expression control sequence; 
 II) a vector comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as  Ostreococcus lucimarinus  CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and 
 wherein the polynucleotide is operatively linked to an expression control sequence, or 
 III) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as  Ostreococcus lucimarinus  CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises 
 a. a nucleic acid sequence as shown in SEQ ID NO: 13, 
 b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or 
 c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) 
 and wherein the polynucleotide is operatively linked to an expression control sequence, 
 
 
         
         under conditions which permit the biosynthesis of the substance. 
       
     
     
         3 . The process according to  claim 1 , wherein the substituents R 2  or R 3  in the general formulae I and II are, independently of one another, saturated or unsaturated C 15 -C 22 -alkylcarbonyl. 
     
     
         4 . The process according to  claim 1 , wherein the substituents R 2  or R 3  in the general formulae I and II are, independently of one another, unsaturated C 18 -, C 20 -, or C 22 -alkylcarbonyl with at least two double bonds. 
     
     
         5 . The process according to  claim 1 , wherein the substance of formula I is stearidonic acid, eicosapentaenoic acid, ω3-docosapentaenoic acid, docosahexaenoic acid or mixtures of these. 
     
     
         6 . The process according to  claim 1 , wherein the substance of formula I is stearidonic acid. 
     
     
         7 . The process according to  claim 1 , wherein the transgenic, nonhuman organism is an animal, a plant or a multicellular microorganism. 
     
     
         8 . The process according to  claim 7 , wherein the transgenic plant is an oil crop. 
     
     
         9 . The process according to  claim 8 , wherein the transgenic plant is a  Brassica species.    
     
     
         10 . The process according to  claim 9 , wherein the  Brassica  species is canola. 
     
     
         11 . A process for the production of an oil, lipid or fatty acid composition, according to  claim 1  comprising the further step of formulating the substance as an oil, lipid or fatty acid composition. 
     
     
         12 . The process according to  claim 10 , wherein the oil, lipid or fatty acid composition is formulated further to give a pharmaceutical, a cosmetic product, a foodstuff, a feeding stuff, or a food supplement. 
     
     
         13 . The process according to  claim 1 , wherein the polypeptide encoded by the nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) has the same delta 6 desaturase activity as  Ostreococcus lucimarinus  delta 6 desaturase. 
     
     
         14 . The process according to  claim 1 , wherein the sequence identity to one of the nucleotide sequences of (a) or (b) is determined over the entire length of the sequences to be compared. 
     
     
         15 . The process of  claim 14  which implements the algorithm of Needleman and Wunsch to compare the sequences. 
     
     
         16 . The process according to  claim 2 , wherein the substituents R 2  or R 3  in the general formulae I and II are, independently of one another, saturated or unsaturated C 15 -C 22 -alkylcarbonyl. 
     
     
         17 . The process according to  claim 2 , wherein the substituents R 2  or R 3  in the general formulae I and II are, independently of one another, unsaturated C 18 -, C 20 -, or C 22 -alkylcarbonyl with at least two double bonds. 
     
     
         18 . The process according to  claim 2 , wherein the substance of formula I is stearidonic acid, eicosapentaenoic acid, ω3-docosapentaenoic acid, docosahexaenoic acid or mixtures of these. 
     
     
         19 . The process according to  claim 2 , wherein the substance of formula I is stearidonic acid. 
     
     
         20 . The process according to  claim 2 , wherein the transgenic, nonhuman organism is an animal, a plant or a multicellular microorganism. 
     
     
         21 . The process according to  claim 20 , wherein the transgenic plant is an oil crop. 
     
     
         22 . The process according to  claim 21 , wherein the transgenic plant is a  Brassica  species. 
     
     
         23 . The process according to  claim 22 , wherein the  Brassica  species is canola. 
     
     
         24 . A process for the production of an oil, lipid or fatty acid composition, according to  claim 2  comprising the further step of formulating the substance as an oil, lipid or fatty acid composition. 
     
     
         25 . The process according to  claim 23 , wherein the oil, lipid or fatty acid composition is formulated further to give a pharmaceutical, a cosmetic product, a foodstuff, a feeding stuff, or a food supplement. 
     
     
         26 . The process according to  claim 2 , wherein the polypeptide encoded by the nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) has the same delta 6 desaturase activity as  Ostreococcus lucimarinus  delta 6 desaturase. 
     
     
         27 . The process according to  claim 2 , wherein the sequence identity to one of the nucleotide sequences of (a) or (b is determined over the entire length of the sequences to be compared. 
     
     
         28 . The process of  claim 27  which implements the algorithm of Needleman and Wunsch to compare the sequences.