Diacylglycerol acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous organisms

Abstract

Acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g., Yarrowia lipolytica ). Specifically, genes encoding diacylglycerol acyltransferase (DGAT1) have been isolated from Y. lipolytica and Mortierella alpina . These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.

1 - 4 . (canceled)  
     
     
         5 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a diacylglycerol acyltransferase-1 enzyme of at least 526 amino acids that has at least 70% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:14; 
 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.    
     
     
         6 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a diacylglycerol acyltransferase-1 enzyme of at least 525 amino acids that has at least 70% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:18; 
 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.    
     
     
         7 . An isolated nucleic acid molecule encoding an acyl-CoA:sterol-acyltransferase, selected from the group consisting of: 
 (a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:16;    (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or    (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).    
     
     
         8 . The isolated nucleic acid molecule of  claim 7  as set forth in SEQ ID NO:15.  
     
     
         9 - 10 . (canceled)  
     
     
         11 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding an acyl-CoA:sterol-acyltransferase enzyme of at least 543 amino acids that has at least 70% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:16; 
 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.    
     
     
         12 - 18 . (canceled)  
     
     
         19 . A method of obtaining a nucleic acid molecule encoding a diacylglycerol acyltransferase-1 enzyme comprising: 
 a) probing a genomic library with a nucleic acid molecule selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:17;    b) identifying a DNA clone that hybridizes with the nucleic acid molecule of (a); and    c) sequencing the genomic fragment that comprises the clone identified in step (b);    wherein the sequenced genomic fragment encodes a diacylglycerol acyltransferase-1 enzyme.    
     
     
         20 . A method of obtaining a nucleic acid molecule encoding a diacylglycerol acyltransferase-1 enzyme comprising: 
 a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NOs:13 and 17; and    b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);    wherein the amplified insert encodes a portion of an amino acid sequence encoding a diacylglycerol acyltransferase 1.    
     
     
         21 . The product of the method of claims  19  or  20 .  
     
     
         22 . An isolated nucleic acid molecule encoding an amino acid motif selected from the group consisting of: 
 a) SEQ ID NO:31;    b) SEQ ID NO:32;    c) SEQ ID NO:33;    d) SEQ ID NO:34;    e) SEQ ID NO:35;    f) SEQ ID NO:36;    g) SEQ ID NO:37;    h) SEQ ID NO:23;    i) SEQ ID NO:24;    j) SEQ ID NO:25;    k) SEQ ID NO:26;    l) SEQ ID NO:27;    m) SEQ ID NO:28;    n) SEQ ID NO:29; and    o) SEQ ID NO:30.    
     
     
         23 . An amino acid motif sequence selected from the group consisting of: 
 a) SEQ ID NO:31;    b) SEQ ID NO:32;    c) SEQ ID NO:33;    d) SEQ ID NO:34;    e) SEQ ID NO:35;    f) SEQ ID NO:36;    g) SEQ ID NO:37;    h) SEQ ID NO:23;    i) SEQ ID NO:24;    j) SEQ ID NO:25;    k) SEQ ID NO:26;    l) SEQ ID NO:27;    m) SEQ ID NO:28;    n) SEQ ID NO:29; and    o) SEQ ID NO:30.    
     
     
         24 . A method of increasing triacylglycerol content in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) at least one gene encoding a diacylglycerol acyltransferase-1 enzyme having the amino acid sequence selected from the group consisting of SEQ ID NOs:14, 18, 19, 20, 21 and 22 under the control of suitable regulatory sequences; and,  
 (ii) a source of fatty acids;  
   (b) growing the cell of step (a) under conditions whereby the at least one gene encoding a diacylglycerol acyltransferase 1 enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and    (c) optionally recovering the triacylglycerol of step (b).    
     
     
         25 . A method of increasing the ω-3 or ω-6 fatty acid content of triacylglycerols in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway;  
 (ii) at least one gene encoding a diacylglycerol acyltransferase 1 enzyme having the amino acid sequence selected from the group consisting of SEQ ID NOs:14, 18, 19, 20, 21 and 22 under the control of suitable regulatory sequences;  
 (b) growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed, resulting in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and  
 (c) optionally recovering the triacylglycerol of step (b).  
   
     
     
         26 . A method of increasing triacylglycerol content in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) at least one gene encoding a diacylglycerol acyltransferase-1 enzyme comprising all of the amino acid motifs as set forth in: 
 1) SEQ ID NO:31;  
 2) SEQ ID NO:32;  
 3) SEQ ID NO:33;  
 4) SEQ ID NO:34;  
 5) SEQ ID NO:35;  
 6) SEQ ID NO:36; and  
 7) SEQ ID NO:37;  
 
 under the control of suitable regulatory sequences; and,  
 (ii) a source of fatty acids;  
   (b) growing the cell of step (a) under conditions whereby the at least one gene encoding a diacylglycerol acyltransferase-1 enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and    (c) optionally recovering the triacylglycerol of step (b).    
     
     
         27 . A method of increasing triacylglycerol content in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) at least one gene encoding a diacylglycerol acyltransferase 1 enzyme comprising all of the amino acid motifs as set forth in: 
 1) SEQ ID NO:23;  
 2) SEQ ID NO:24;  
 3) SEQ ID NO:25;  
 4) SEQ ID NO:26;  
 5) SEQ ID NO:27;  
 6) SEQ ID NO:28;  
 7) SEQ ID NO:29; and  
 8) SEQ ID NO:30;  
 
 under the control of suitable regulatory sequences; and,  
 (ii) a source of fatty acids;  
   (b) growing the cell of step (a) under conditions whereby the at least one gene encoding a diacylglycerol acyltransferase-1 enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and    (c) optionally recovering the triacylglycerol of step (b).    
     
     
         28 . A method of increasing the ω-3 or ω-6 fatty acid content of triacylglycerols in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway; and  
 (ii) at least one gene encoding a diacylglycerol acyltransferase 1 enzyme comprising all of the amino acid motifs as set forth in: 
 1) SEQ ID NO:31;  
 2) SEQ ID NO:32;  
 3) SEQ ID NO:33;  
 4) SEQ ID NO:34;  
 5) SEQ ID NO:35;  
 6) SEQ ID NO:36; and  
 7) SEQ ID NO:37;  
 
 under the control of suitable regulatory sequences;  
   (b) growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed, resulting in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and    (c) optionally recovering the triacylglycerol of step (b).    
     
     
         29 . A method of increasing the ω-3 or ω-6 fatty acid content of triacylglycerols in a transformed host cell comprising: 
 (a) providing a transformed host cell comprising: 
 (i) genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway; and  
 (ii) at least one gene encoding a diacylglycerol acyltransferase 1 enzyme comprising all of the amino acid motifs as set forth in: 
 1) SEQ ID NO:23;  
 2) SEQ ID NO:24;  
 3) SEQ ID NO:25;  
 4) SEQ ID NO:26;  
 5) SEQ ID NO:27;  
 6) SEQ ID NO:28;  
 7) SEQ ID NO:29; and  
 8) SEQ ID NO:30;  
 
 under the control of suitable regulatory sequences;  
   (b) growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed, resulting in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol; and    (c) optionally recovering the triacylglycerol of step (b).    
     
     
         30 . A method according to  claim 25 ,  28  or  29 , wherein the genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway are selected from the group consisting of desaturases and elongases.  
     
     
         31 . A method according to  claim 30 , wherein the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, a Δ8 desaturase, Δ15 desaturase and Δ4 desaturase.  
     
     
         32 . A method according to any of claims  24 - 29 , wherein the host cell is selected from the group consisting of algae, bacteria, molds, fungi and yeasts.  
     
     
         33 . A method according to  claim 32 , wherein the host cell is an oleaginous yeast.  
     
     
         34 . A method according to  claim 33  wherein the oleaginous yeast is a member of a genus selected from the group of consisting of  Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon  and  Lipomyces.    
     
     
         35 . A method according to  claim 34 , wherein the oleaginous yeast is  Yarrowia lipolytica.    
     
     
         36 . A method according to  claim 35 , wherein the  Yarrowia lipolytica  is a strain selected from the group consisting of  Yarrowia lipolytica  ATCC #20362,  Yarrowia lipolytica  ATCC #8862,  Yarrowia lipolytica  ATCC #18944,  Yarrowia lipolytica  ATCC #76982,  Yarrowia lipolytica  ATCC #90812 and  Yarrowia lipolytica  LGAM S(7)1.  
     
     
         37 . A method according to any one of claims  24 ,  25 ,  26 ,  27 ,  28  or  29 , wherein the fatty acid is selected from the group consisting of: stearate, oleic acid, linoleic acid, γ-linoleic acid, dihomo-γ-linoleic acid, arachidonic acid, α-linoleic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, eicosadienoic acid and eicosatrienoic acid.  
     
     
         38 . A method for the identification of a polypeptide having diacylglycerol acyltransferase-1 activity comprising: 
 a) obtaining the amino acid sequence of a polypeptide suspected of having diacylglycerol acyltransferase-1 activity; and,    b) identifying, in the amino acid sequence of the polypeptide of step (a), the presence of all of the amino acid motif sequences as set forth in: 
 1) SEQ ID NO:31;  
 2) SEQ ID NO:32;  
 3) SEQ ID NO:33;  
 4) SEQ ID NO:34;  
 5) SEQ ID NO:35;  
 6) SEQ ID NO:36; and  
 7) SEQ ID NO:37;  
 wherein the presence of all of the motif sequences of step (a) in the polypeptide is indicative of diacylglycerol acyltransferase-1 activity.  
   
     
     
         39 . A method for the identification of a fungal polypeptide having diacylglycerol acyltransferase-1 activity comprising: 
 a) obtaining the amino acid sequence of a fungal polypeptide suspected of having diacylglycerol acyltransferase-1 activity; and,    b) identifying, in the amino acid sequence of the polypeptide of step (a), the presence of all of the amino acid motif sequences as set forth in: 
 1) SEQ ID NO:23;  
 2) SEQ ID NO:24;  
 3) SEQ ID NO:25;  
 4) SEQ ID NO:26;  
 5) SEQ ID NO:27;  
 6) SEQ ID NO:28;  
 7) SEQ ID NO:29; and  
 8) SEQ ID NO:30;  
 wherein the presence of all of the motif sequences of step (a) in the polypeptide is indicative of diacylglycerol acyltransferase-1 activity.