Schizochytrium Fatty Acid Synthase (FAS) and Products and Methods Related Thereto

Abstract

Disclosed are a fatty acid synthase (FAS) from Schizochytrium , biologically active fragments and homologues thereof, a nucleic acid sequence encoding such FAS, fragments and homologues thereof, the gene encoding Schizochytrium FAS, host cells and organisms that recombinantly express the FAS, host cells and organisms in which the expression and/or activity of the endogenous FAS has been attenuated, and various methods for making and using any of these proteins, nucleic acid molecules, genes, host cells or organisms.

1 . 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:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof; and   b) an amino acid sequence that is at least about 45% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a).   
     
     
         2 . The isolated protein of  claim 1 , wherein the isolated protein comprises an amino acid sequence that is at least about 60% identical to any of the amino acid sequences of (a). 
     
     
         3 . The isolated protein of  claim 1 , wherein the protein comprises an amino acid sequence that is at least about 80% identical to any of the amino acid sequences of (a). 
     
     
         4 . The isolated protein of  claim 1 , wherein the protein comprises an amino acid sequence that is at least about 95% identical to any of the amino acid sequences of (a). 
     
     
         5 . The isolated protein of  claim 1 , wherein the protein comprises an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, or biologically active fragments of any of these sequences. 
     
     
         6 . The isolated protein of  claim 1 , wherein the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. 
     
     
         7 . The isolated protein of  claim 1 , wherein the protein comprises any two or more amino acid sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. 
     
     
         8 . The isolated protein of  claim 1 , wherein the protein comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. 
     
     
         9 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the protein of  claim 1 , or a nucleic acid sequence that is fully complementary to the nucleic acid sequence of  claim 1 . 
     
     
         10 . A recombinant nucleic acid molecule comprising an isolated nucleic acid molecule as set forth in  claim 9 , operatively linked to a transcription control sequence. 
     
     
         11 . The recombinant nucleic acid molecule of  claim 10 , wherein the transcription control sequence is a tissue-specific transcription control sequence. 
     
     
         12 . The recombinant nucleic acid molecule of  claim 11 , further comprising a targeting sequence. 
     
     
         13 . A recombinant cell that has been transformed with the recombinant nucleic acid molecule of  claim 10 . 
     
     
         14 . A genetically modified microorganism for producing short chain fatty acids by a biosynthetic process, the microorganism being transformed with a recombinant nucleic acid molecule according to  claim 10 . 
     
     
         15 . A genetically modified plant for producing short chain fatty acids by a biosynthetic process, the plant being transformed with a recombinant nucleic acid molecule according to  claim 10 . 
     
     
         16 . A genetically modified microorganism for producing short chain fatty acids by a biosynthetic process, wherein the microorganism comprises a nucleic acid molecule encoding a fatty acid synthase and wherein the nucleic acid molecule encoding the fatty acid synthase has been modified to increase the expression or biological activity of the fatty acid synthase, the fatty acid synthase 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:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, an amino acid sequence consisting of positions 1-500 of SEQ ID NO:2, an amino acid sequence consisting of positions 450-1300 of SEQ ID NO:2, an amino acid sequence consisting of positions 1250-1700 of SEQ ID NO:2, an amino acid sequence consisting of positions 1575-2100 of SEQ ID NO:2, an amino acid sequence consisting of positions 2025-2850 of SEQ ID NO:2, an amino acid sequence consisting of positions 2800-3350 of SEQ ID NO:2, an amino acid sequence consisting of positions 3300-3900 of SEQ ID NO:2, an amino acid sequence consisting of positions 3900-4136 of SEQ ID NO:2, and a biologically active fragment thereof; and b) an amino acid sequence that is at least about 45% identical to any of the amino acid sequences of (a) and having the biological activity of the amino acid sequence of (a). 
     
     
         17 . The genetically modified microorganism of  claim 16 , wherein the nucleic acid molecule encoding a fatty acid synthase is an endogenous gene in the microorganism. 
     
     
         18 . The genetically modified microorganism of  claim 16 , wherein the microorganism has been transformed with a nucleic acid molecule encoding the fatty acid synthase. 
     
     
         19 . The genetically modified microorganism of  claim 16 , wherein the microorganism comprises an endogenous gene encoding the fatty acid synthase and has been transformed with a recombinant nucleic acid molecule encoding a fatty acid synthase, wherein one or both of the gene and the recombinant nucleic acid molecule has been modified to increase the expression or biological activity of the fatty acid synthase. 
     
     
         20 . The genetically modified microorganism of  claim 16 , wherein the microorganism is a Thraustochytriales microorganism. 
     
     
         21 . A biomass comprising the genetically modified microorganism of  claim 16 . 
     
     
         22 . A food product or a pharmaceutical product comprising the biomass of  claim 21 . 
     
     
         23 . A method to produce short chain fatty acids by a biosynthetic process, comprising culturing in a fermentation medium a genetically modified microorganism according to  claim 16 . 
     
     
         24 . A method to produce short chain fatty acids by a biosynthetic process, comprising growing a genetically modified plant that has been transformed with a recombinant nucleic acid molecule as set forth in  claim 10 . 
     
     
         25 . An oligonucleotide, comprising at least 12 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, or a nucleic acid sequence fully complementary thereto. 
     
     
         26 . A genetically modified microorganism with reduced production of short chain fatty acids, wherein the microorganism has been genetically modified to selectively attenuate a fatty acid synthase gene or portion thereof encoding a functional domain, wherein the fatty acid synthase gene comprises a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence encoding SEQ ID NO:2; and b) a nucleic acid sequence encoding an amino acid sequence that is at least about 45% identical to SEQ ID NO:2, wherein the protein having the amino acid sequence has a biological activity selected from the group consisting of acetyl-transferase (AT) activity; enoyl ACP reductase (ER) activity; dehydrase (DH) activity; malonyl/palmitoyl acyltransferase (M/PAT) activity; a first acyl carrier protein (ACP) activity; a second acyl carrier protein (ACP) activity; keto-acyl ACP reductase (KR) activity; keto-acyl ACP synthase (KS) activity; and phosphopantetheinyl transferase (PPT) activity. 
     
     
         27 . The genetically modified microorganism of  claim 26 , wherein the fatty acid synthase gene comprises a nucleic acid sequence represented by SEQ ID NO:1. 
     
     
         28 . The genetically modified microorganism of  claim 26 , wherein the microorganism has increased production of at least one polyunsaturated fatty acid (PUFA). 
     
     
         29 . The genetically modified microorganism of  claim 26 , wherein the microorganism is a Thraustochytriales microorganism. 
     
     
         30 . The genetically modified microorganism of  claim 26 , wherein the fatty acid synthase gene has been modified in a regulatory region to reduce expression of the gene. 
     
     
         31 . The genetically modified microorganism of  claim 26 , wherein the fatty acid synthase gene has been modified in the coding region to reduce the biological activity of one or more functional domains of the fatty acid synthase. 
     
     
         32 . The genetically modified microorganism of  claim 26 , wherein the fatty acid synthase gene has been mutated by targeted homologous recombination with a nucleic acid sequence that hybridizes to the fatty acid synthase gene and includes a heterologous nucleic acid sequence that modifies the coding region of the fatty acid synthase gene to reduce the expression or activity of the fatty acid synthase encoded thereby. 
     
     
         33 . A biomass comprising genetically modified microorganisms as set forth in  claim 26 , wherein the microorganisms have reduced production of short chain fatty acids as compared to a wild-type microorganism of the same species. 
     
     
         34 . A food product or a pharmaceutical product comprising the biomass according to  claim 33 . 
     
     
         35 . A method for increasing the production of polyunsaturated fatty acids (PUFAs) in a biosynthetic process, comprising culturing under conditions effective to produce lipids comprising the PUFAs, genetically modified microorganisms according to  claim 26 . 
     
     
         36 . A product comprising the lipids produced by the method of  claim 35 .