Mutant Delta-8 Desaturase Genes Engineered By Targeted Mutagensis and Their Use In Making Polyunsaturated Fatty Acids

Abstract

The present invention relates to mutant delta-8 desaturase genes, which have the ability to convert eicosadienoic acid [20:2 omega-6, EDA] to dihomo-γ-linolenic acid [20:3, DGLA] and/or eicosatrienoic acid [20:3 omega-3, ETrA] to eicosatetraenoic acid [20:3 omega-3, ETA]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-8 desaturase along with methods of making long-chain polyunsaturated fatty acids (PUFAs) using these mutant delta-8 desaturases in plants and oleaginous yeast are disclosed.

1 - 22 . (canceled) 
     
     
         23 . Oil obtained from a transgenic seed obtained from an oilseed plant comprising a recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide comprising:
 (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:2, wherein SEQ ID NO:2 comprises at least one mutation selected from the group consisting of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10;   (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:198, wherein SEQ ID NO:198 comprises at least one mutation selected from the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E, V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S, Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, F297V, V298L, 1309V, V310I, I346V, I347L, T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; or (c) a complement of the nucleotide sequence of part (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         24 . The oil of  claim 23 , wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax, and safflower. 
     
     
         25 . Oil obtained from a transgenic seed obtained from an oilseed plant comprising:
 (a) a first recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in
 (i) SEQ ID NO:2, wherein SEQ ID NO:2 comprises at least one mutation selected from the group consisting of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; or 
 (ii) SEQ ID NO:198, wherein SEQ ID NO:198 comprises at least one mutation selected from the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E, V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V1225, Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, F297V, V298L, I309V, V310I, I346V, I347L, T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; and 
   (b) at least one additional recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C 14/18  elongase, a C 16/18  elongase, a C 18/20  elongase, and a C 20/22  elongase.   
     
     
         26 . The oil of  claim 25 , wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax, and safflower. 
     
     
         27 . Food or feed which incorporates the oil of any of  claims 23 - 26 . 
     
     
         28 . Food or feed comprising an ingredient derived from the processing of a transgenic seed obtained from an oilseed plant comprising a recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide comprising:
 (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:2, wherein SEQ ID NO:2 comprises at least one mutation selected from the group consisting of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10;   (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO:198, wherein SEQ ID NO:198 comprises at least one mutation selected from the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E, V20I, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S, Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, F297V, V298L, I309V, V310I, I346V, I347L, T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; or   (c) a complement of the nucleotide sequence of part (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         29 . The food or feed of  claim 28 , wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax, and safflower. 
     
     
         30 . Food or feed comprising an ingredient derived from the processing of a transgenic seed obtained from an oilseed plant comprising:
 (a) a first recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity wherein the polypeptide has an amino acid sequence as set forth in
 (i) SEQ ID NO:2, wherein SEQ ID NO:2 comprises at least one mutation selected from the group consisting of S4A, K5S, T12V, T16K, T17V, A54G, F55Y, P66Q, S67A, S108L, G117A, Y118F, L120M, M121L, Q125H, M126L, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, I346V, I347L, T348S, A407S, V408Q, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; or 
 (ii) SEQ ID NO:198, wherein SEQ ID NO:198 comprises at least one mutation selected from the group consisting of S4A, K5S, L9V, T12V, T16K, T17V, D19E, V201, A54G, F55Y, K59L, I64L, N65D, N65Q, P66Q, S67A, E69D, L70V, A75G, V76L, S108L, G117A, Y118F, L120M, M121L, V122S, Q125H, M126L, Y127Q, V132L, L133V, L162V, V163L, G170A, L171V, T279L, L280T, L293M, F297V, V298L, I309V, V310I, I346V, I347L, T348S, A407S, V408Q, Q416V, P417Y, A418G, G419A, and L422Q, wherein the mutations are defined with respect to SEQ ID NO:10; and 
   (b) at least one additional recombinant construct comprising at least one regulatory sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C 14/16  elongase, a C 16/18  elongase, a C 18/20  elongase, and a C 20/22  elongase.   
     
     
         31 . The food or feed of  claim 30 , wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax, and safflower.