Pufa polyketide synthase systems and uses thereof

Abstract

The invention generally relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems, to homologues thereof, to isolated nucleic acid molecules and recombinant nucleic acid molecules encoding biologically active domains of such a PUFA PKS system, to genetically modified organisms comprising PUFA PKS systems, to methods of making and using such systems for the production of bioactive molecules of interest, and to novel methods for identifying new bacterial and non-bacterial microorganisms having such a PUFA PKS system.

1 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and biologically active fragments thereof;   (b) a nucleic acid sequence encoding an amino acid sequence that is at least about 60% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:56 and SEQ ID NO:58, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system;   (c) a nucleic acid sequence encoding an amino acid sequence that is at least about 65% identical to SEQ ID NO:54, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system;   (d) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system;   (e) a nucleic acid sequence encoding an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:66, SEQ ID NO:68, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; and   (f) a nucleic acid sequence that is fully complementary to the nucleic acid sequence of (a), (b), (c), (d), or (e).   
     
     
         2 .- 4 . (canceled) 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and biologically active fragments thereof. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence is selected from the group consisting of: SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, and SEQ ID NO:67. 
     
     
         7 . A recombinant nucleic acid molecule comprising the nucleic acid molecule of  claim 1 , operatively linked to at least one transcription control sequence. 
     
     
         8 . A recombinant cell transfected with the recombinant nucleic acid molecule of  claim 7 . 
     
     
         9 . A genetically modified microorganism, wherein the microorganism expresses a PKS system comprising at least one biologically active domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, wherein the microorganism comprises the nucleic acid molecule of  claim 1 . 
     
     
         10 .- 23 . (canceled) 
     
     
         24 . The genetically modified microorganism of  claim 9 , wherein the microorganism endogenously expresses a PUFA PKS system comprising the at least one biologically active domain of a PUFA PKS system, and wherein the genetic modification comprises expression of a recombinant nucleic acid molecule selected from the group consisting of a recombinant nucleic acid molecule encoding at least one biologically active domain from a second PKS system and a recombinant nucleic acid molecule encoding a protein that affects the activity of the endogenous PUFA PKS system. 
     
     
         25 . (canceled) 
     
     
         26 . The genetically modified microorganism of  claim 24 , wherein the recombinant nucleic acid molecule encodes a phosphopantetheine transferase. 
     
     
         27 .- 30 . (canceled) 
     
     
         31 . A genetically modified plant, wherein the plant has been genetically modified to recombinantly express a PKS system comprising at least one biologically active domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, wherein the plant comprises the nucleic acid molecule of  claim 1 . 
     
     
         32 .- 38 . (canceled) 
     
     
         39 . A method to produce a bioactive molecule that is produced by a polyketide synthase system, comprising culturing under conditions effective to produce the bioactive molecule a genetically modified organism comprising the nucleic acid molecule of  claim 1 . 
     
     
         40 .- 54 . (canceled) 
     
     
         55 . A method to produce a plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the naturally occurring plant, comprising genetically modifying cells of the plant to express a PKS system comprising at least one recombinant nucleic acid molecule comprising the nucleic acid molecule of  claim 1 . 
     
     
         56 . A method to modify an endproduct containing at least one fatty acid, comprising adding to the endproduct an oil produced by a recombinant host cell that expresses at least one recombinant nucleic acid molecule comprising the nucleic acid molecule of  claim 1 . 
     
     
         57 .- 59 . (canceled) 
     
     
         60 . A method to produce a humanized animal milk, comprising genetically modifying milk-producing cells of a milk-producing animal with at least one recombinant nucleic acid molecule comprising the nucleic acid molecule of  claim 1 . 
     
     
         61 . A genetically modified Thraustochytrid microorganism, wherein the microorganism has an endogenous polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, and wherein the endogenous PUFA PKS system has been genetically modified to alter the expression profile of a polyunsaturated fatty acid (PUFA) by the Thraustochytrid microorganism as compared to the Thraustochytrid microorganism in the absence of the genetic modification. 
     
     
         62 . (canceled) 
     
     
         63 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the endogenous PUFA PKS system has been modified by deleting at least one nucleic acid sequence that encodes at least one domain of the endogenous PUFA PKS system and inserting therefore a nucleic acid sequence encoding a homologue of the endogenous domain to alter the PUFA production profile of the Thraustochytrid microorganism, wherein the homologue has a biological activity of at least one domain of a PKS system. 
     
     
         64 . (canceled) 
     
     
         65 . The genetically modified Thraustochytrid microorganism of  claim 63 , wherein the domain of the endogenous PUFA PKS system is a domain having a biological activity of at least one of the following proteins: malonyl-CoA:ACP acyltransferase (MAT), β-keto acyl-ACP synthase (KS), ketoreductase (KR), acyltransferase (AT), FabA-like β-hydroxy acyl-ACP dehydrase (DH), phosphopantetheine transferase, chain length factor (CLF), acyl carrier protein (ACP), enoyl ACP-reductase (ER), an enzyme that catalyzes the synthesis of trans-2-acyl-ACP, an enzyme that catalyzes the reversible isomerization of trans-2-acyl-ACP to cis-3-acyl-ACP, and an enzyme that catalyzes the elongation of cis-3-acyl-ACP to cis-5-β-keto-acyl-ACP. 
     
     
         66 .- 72 . (canceled) 
     
     
         73 . The genetically modified Thraustochytrid microorganism of  claim 63 , wherein the homologue of the endogenous domain comprises a modification, as compared to the endogenous domain, selected from the group consisting of at least one deletion, insertion or substitution that results in an alteration of PUFA production profile by the microorganism. 
     
     
         74 .- 82 . (canceled) 
     
     
         83 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the PUFA production profile is altered to initiate, increase or decrease production of eicosapentaenoic acid (EPA) by the microorganism. 
     
     
         84 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the PUFA production profile is altered to initiate, increase or decrease production of docosahexaenoic acid (DHA) by the microorganism. 
     
     
         85 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the PUFA production profile is altered to initiate, increase or decrease production of one or both isomers of docosapentaenoic acid (DPA) by the microorganism. 
     
     
         86 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the PUFA production profile is altered to initiate, increase or decrease production of arachidonic acid (ARA) by the microorganism. 
     
     
         87 .- 90 . (canceled) 
     
     
         91 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the microorganism is a  Schizochytrium  that produces eicosapentaenoic acid (EPA), wherein the  Schizochytrium  has an endogenous polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system comprising a genetic modification in at least one nucleic acid sequence that encodes at least one domain of the endogenous PUFA PKS system that results in the production of EPA by the  Schizochytrium.    
     
     
         92 .- 108 . (canceled) 
     
     
         109 . The genetically modified Thraustochytrid microorganism of  claim 61 , wherein the microorganism is a  Schizochytrium  that produces increased amounts of docosahexaenoic acid (DHA) as compared to a non-genetically modified  Schizochytrium , wherein the  Schizochytrium  has an endogenous polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system comprising a genetic modification in at least one nucleic sequence that encodes at least one domain of the endogenous PUFA PKS system that results in increased the production of DHA by the  Schizochytrium.    
     
     
         110 . The genetically modified  Schizochytrium  of  claim 109 , wherein at least one domain of the endogenous PUFA PKS system has been modified by substitution for at least one domain of a PUFA PKS system from  Thraustochytrium.    
     
     
         111 . The genetically modified  Schizochytrium  of  claim 109 , wherein the ratio of DHA to DPA produced by the  Schizochytrium  is increased as compared to a non-genetically modified  Schizochytrium.    
     
     
         112 . A method to produce lipids enriched for at least one selected polyunsaturated fatty acid (PUFA), comprising culturing under conditions effective to produce the lipids a genetically modified  Schizochytrium  according to  claim 109 . 
     
     
         113 . (canceled) 
     
     
         114 . A method to produce eicosapentaenoic acid (EPA)-enriched lipids, comprising culturing under conditions effective to produce the EPA-enriched lipids the genetically modified Thraustochytrid microorganism of  claim 61 , wherein the microorganism has an endogenous polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system, and wherein the endogenous PUFA PKS system has been genetically modified in at least one domain to initiate or increase the production of EPA in the lipids of the microorganism as compared to in the absence of the modification. 
     
     
         115 . An isolated protein comprising an amino acid sequence selected from the group consisting of:
 (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and biologically active fragments thereof;   (b) an amino acid sequence that is at least about 60% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:56 and SEQ ID NO:58, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system;   (c) an amino acid sequence that is at least about 65% identical to SEQ ID NO:54, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system;   (d) an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; and   (e) an amino acid sequence that is at least about 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:66, SEQ ID NO:68, wherein the amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system.