Oleic acid production in yeast

Abstract

Disclosed are transformed cells comprising one or more genetic modifications that affect the lipid content of the cell, e.g., by increasing the concentration of oleic acid in the cell relative to an unmodified cell of the same type. Also disclosed are methods for modifying the lipid content of a cell by increasing the activity of one or more proteins in the cell and/or by decreasing the activity of one or more proteins in the same cell.

1 - 20 . (canceled) 
     
     
         21 . A method for producing a product, wherein the product is a lipid, the method comprising cultivating a modified yeast cell thereby producing the lipid, wherein the modified yeast cell comprises:
 a first genetic modification, wherein the first genetic modification comprises a knockout mutation of a native Δ12 desaturase protein;   a second genetic modification, wherein the second genetic modification increases the expression of an elongase protein, a diacylglycerol acyltransferase protein, or a glycerol-3-phosphate acyltransferase protein; and   a third genetic modification, wherein the third genetic modification comprises a nucleic acid encoding an exogenous fungal Δ9 desaturase protein or an exogenous Δ9 desaturase protein that comprises increased specificity for C18 a fatty acid relative to a native Δ9 desaturase protein of the modified yeast cell.   
     
     
         22 . The method of  claim 21 , wherein the second genetic modification comprises a nucleic acid encoding an elongase protein, a diacylglycerol acyltransferase protein, or a glycerol-3-phosphate acyltransferase protein. 
     
     
         23 . The method of  claim 21 , wherein the exogenous fungal Δ9 desaturase protein is a  Puccinia graminis, Arxula adeninivorans  or  Microbotryum violaceum  Δ9 desaturase protein. 
     
     
         24 . The method of  claim 21 , wherein the yeast cell is selected from the group consisting of  Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces , and  Yarrowia.    
     
     
         25 . The method of  claim 21 , wherein the yeast cell is selected from the group consisting of  Arxula adeninivorans, Saccharomyces cerevisiae , and  Yarrowia lipolytica.    
     
     
         26 . The method of  claim 21 , wherein the yeast cell comprises at least 50% lipid as measured by % dry cell weight. 
     
     
         27 . The method of  claim 21 , wherein the yeast cell comprises oleic acid at a concentration of at least 70% as a percentage of total C16 and C18 fatty acids in the yeast cell. 
     
     
         28 . The method of  claim 21 , wherein the second genetic modification increases the expression of a glycerol-3-phosphate acyltransferase protein. 
     
     
         29 . The method of  claim 28 , wherein the glycerol-3-phosphate acyltransferase protein is a native protein. 
     
     
         30 . The method of  claim 22 , wherein the second genetic modification comprises a nucleic acid encoding an exogenous glycerol-3-phosphate acyltransferase protein. 
     
     
         31 . The method of  claim 21 , wherein the second genetic modification increases the expression of an elongase protein. 
     
     
         32 . The method of  claim 21 , wherein the second genetic modification increases the expression of a diacylglycerol acyltransferase protein. 
     
     
         33 . The method of  claim 21 , wherein the yeast cell comprises a knockout mutation of a native Δ9 desaturase protein. 
     
     
         34 . The method of  claim 22 , wherein the glycerol-3-phosphate acyltransferase protein comprises an  Arxula adeninivorans  glycerol-3-phosphate acyltransferase, a  Saccharomyces cerevisiae  glycerol-3-phosphate acyltransferase, or a glycerol-3-phosphate acyltransferase comprising amino acid sequence having 90% sequence identity to the sequence encoded by SEQ ID NO: 44 or 42. 
     
     
         35 . The method of  claim 22 , wherein the elongase protein is an exogenous elongase protein. 
     
     
         36 . The method of  claim 22 , wherein the elongase protein comprises a  Rattus norvegicus  elongase, a  Yarrowia lipolytica  elongase, an  Arxula Adeninivorans  elongase, or an elongase comprising an amino acid sequence having 90% sequence identity to the sequence encoded by SEQ ID NO: 156, 6, or 108. 
     
     
         37 . The method of  claim 22 , wherein the diacylglycerol acyltransferase protein is an exogenous diacylglycerol acyltransferase protein. 
     
     
         38 . The method of  claim 22 , wherein the diacylglycerol acyltransferase protein comprises a  Rhodosporidium toruloides  diacylglycerol acyltransferase, a  Yarrowia lipolytica  diacylglycerol acyltransferase, an  Arxula adeninivorans  diacylglycerol acyltransferase, a  Claviceps  purpurea diacylglycerol acyltransferase or a diacylglycerol acyltransferase comprising an amino acid sequence having 90% sequence identity to the sequence set forth in SEQ ID NO: 22, 20, 52, or 38. 
     
     
         39 . The method of  claim 21 , wherein the exogenous Δ9 desaturase protein is a fungal Δ9 desaturase protein. 
     
     
         40 . The method of  claim 21 , wherein the Δ9 desaturase protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 14, 8 or 12. 
     
     
         41 . The method of  claim 21 , wherein the second genetic modification comprises a nucleic acid encoding (a) a diacylglycerol acyltransferase type 1 protein, (b) a diacylglycerol acyltransferase type 2 protein, or (c) a diacylglycerol acyltransferase type 1 protein and a diacylglycerol acyltransferase type 2 protein. 
     
     
         42 . The method of  claim 21 , wherein the second genetic modification comprises one or more nucleic acids encoding an elongase protein, a diacylglycerol acyltransferase protein, and a glycerol-3-phosphate acyltransferase protein. 
     
     
         43 . The method of  claim 42 , wherein (a) the elongase protein comprises a  Rattus norvegicus  elongase, a  Yarrowia lipolytica  elongase, or an elongase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 156, 6 or 108; (b) the diacylglycerol acyltransferase protein comprises a  Rhodosporidium toruloides  diacylglycerol acyltransferase, a  Yarrowia lipolytica  diacylglycerol acyltransferase, an  Arxula adeninivorans  diacylglycerol acyltransferase, a  Claviceps purpurea  diacylglycerol acyltransferase, or a diacylglycerol acyltransferase comprising an amino acid sequence having 90% sequence identity to the amino acid sequence set forth in SEQ ID Nos. 22, 20, 52, or 38; and (c) the glycerol-3-phosphate acyltransferase protein comprises an  Arxula adeninivorans  glycerol-3-phosphate acyltransferase or a glycerol-3-phosphate acyltransferase comprising an amino acid sequence having 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 44 or 42.