Diacylglycerol acyltransferase (dga1) polynucleotides, and methods of increasing yeast cell lipid production by overexpression of heterologous dga1

Abstract

DGA1 catalyzes the final enzymatic step for converting acyl-CoA and 1,2-diacylglycerol to triacylglycerols (TAG) and CoA in yeast. Disclosed are methods for expression in an oleaginous yeast host of polynucleotide sequences encoding DGA1 from Rhodosporidium toruloides, Lipomyces starkeyi, Aurantiochytrium limacinum, Aspergillus terreus, or Claviceps purpurea. Also described herein are engineered recombinant host cells of Yarrowia lipolytica comprising heterologous DGA1 polynucleotides encoding DGA1 proteins, or functionally active portions thereof, having the capability of producing increased lipid production and possessing the characteristic of enhanced glucose consumption efficiency.

1 . A method for producing a recombinant yeast cell, the method comprising the steps of:
 a) introducing into a yeast cell a recombinant DNA construct comprising a heterologous polynucleotide selected from the group consisting of:
 i. a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, or a complement thereof, and 
 ii. a nucleic acid molecule having at least 80% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, or a complement thereof, and 
   b) expressing a heterologous polypeptide selected from the group consisting of:
 i. an amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or a biologically-active portion thereof, and 
   ii. a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or a biologically-active portion thereof, and   c) cultivating the yeast cell under conditions for increasing lipid production.   
     
     
         2 . The method of  claim 1 , wherein the yeast cell is  Y. lipolytica.    
     
     
         3 . The method of  claim 1 , wherein the polynucleotide is selected from the group consisting of a nucleic acid molecule having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18. 
     
     
         4 . The method of  claim 1 , wherein the polynucleotide is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 6, and SEQ ID NO: 8. 
     
     
         5 . The method of  claim 1 , wherein the polypeptide is selected from the group consisting of a polypeptide having at least 95% sequence identity to the amino acid sequence set forth SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17. 
     
     
         6 . The method of  claim 1 , wherein the polypeptide is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and SEQ ID NO: 7. 
     
     
         7 - 25 . (canceled) 
     
     
         26 . The method of  claim 1 , wherein the recombinant yeast cell is oleaginous, high-temperature tolerant, or both. 
     
     
         27 . The method of  claim 1 , the recombinant yeast cell is grown in the presence of a substrate selected from the group consisting of glucose, ethanol, xylose, sucrose, starch, starch dextrin, glycerol, cellulose, and acetic acid. 
     
     
         28 . The method of  claim 1 , wherein the recombinant yeast cell is an oleaginous yeast cell, and said oleaginous yeast cell is selected from the group consisting of  Rhodosporidium toruloides, Rhodosporidium babjevae, Rhodosporidium paludigenum, Lipomyces starkeyi, Lipomyces tetrasporus, Lipomyces lipofer, Cryptococcus curvatus, Cryptococcus albidus, Cryptococcus terreus, Cryptococcus ramirezgomezianus, Cryptococcus wieringae, Rhodotorula glutinis, Rhodotorula mucilaginosa, Trichosporon cutaneum, Cunninghamella echinulata, Mortierella isabellina, Trichosporon fermentans, Cunninghamella japonica, Aurantiochytrium limacinum, Rhizopus arrhizus, Aspergillus terreus, Claviceps purpurea, Leucosporidiella creatinivora, Tremella enchepala, Yarrowia lipolytica, and Prototheca zopfii.    
     
     
         29 . The method of  claim 28 , wherein the oleaginous yeast cell is  Yarrowia lipolytica.    
     
     
         30 . The method of  claim 1 , wherein the recombinant yeast cell is  Arxula adeniovorans  or  Kluyveromyces marxianus.