Ammonium transporter promoter for gene expression in oleaginous yeast

Abstract

The promoter region associated with the Yarrowia lipolytica ammonium transporter (yat1) gene has been found to be particularly effective for the expression of heterologous genes in oleaginous yeast. The promoter regions of the instant invention have been shown to be advantageously inducible under oleaginous conditions (i.e., nitrogen limitation) and are useful to drive expression of genes involved in the production of ω-3 and ω-6 fatty acids.

1 . A method for the expression of a coding region of interest in a transformed yeast comprising: 
 c) providing a transformed yeast having a genetic construct comprising: 
 (i) a promoter region of a  Yarrowia  yat1 gene; and,  
 (ii) a coding region of interest expressible in the yeast cell;  
   wherein the promoter region is operably linked to the coding region of interest; and,    d) growing the transformed yeast of step (a) under conditions whereby the genetic construct of step (a) is expressed.    
     
     
         2 . A method according to  claim 1  wherein the  Yarrowia  yat1 gene is isolated from  Yarrowia lipolytica.    
     
     
         3 . A method according to  claim 1  wherein the promoter region of the yat1 gene is contained within a nucleic acid molecule as set forth in SEQ ID NO:3.  
     
     
         4 . A method according to  claim 3  wherein the promoter region contains at least one mutation that does not diminish its promoter activity.  
     
     
         5 . A method according to  claim 4  wherein the promoter activity is at least about 20% to at least about 400% of the promoter activity of the wildtype promoter activity.  
     
     
         6 . A method according to  claim 5  wherein the promoter activity is determined by histochemical assay.  
     
     
         7 . A method according to  claim 1  wherein the transformed yeast is an oleaginous yeast.  
     
     
         8 . A method of  claim 7 , wherein the oleaginous yeast is a member of a genus selected from the group consisting of  Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon  and  Lipomyces.    
     
     
         9 . A method according to  claim 8 , wherein the oleaginous yeast is selected from the group consisting of:  Yarrowia lipolytica  ATCC #20362,  Yarrowia lipolytica  ATCC #8862,  Yarrowia lipolytica  ATCC #18944,  Yarrowia lipolytica  ATCC #76982 and  Yarrowia lipolytica  LGAM S(7)1.  
     
     
         10 . A method according to  claim 1  wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases, elongases, acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalyases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, α-galactosidases, β-galactosidases, glucoamylases, α-glucosidases, β-glucanases, β-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases.  
     
     
         11 . A method according to  claim 1  wherein the conditions whereby the genetic construct of step (a) is expressed are those of nitrogen-limitation.  
     
     
         12 . A method according to  claim 11  wherein the conditions of nitrogen-limitation are conditions that induce oleaginy in the transformed yeast.  
     
     
         13 . A method for the production of an ω-3 or an ω-6 fatty acid comprising: 
 d) providing a transformed oleaginous yeast comprising a genetic construct, comprising: 
 (i) a promoter region of a  Yarrowia  yat1 gene; and,  
 (ii) a coding region encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway;  
   wherein the promoter region and coding region are operably linked;    e) culturing the transformed oleaginous yeast of step (a) under conditions whereby the at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is expressed and a ω-3 or ω-6 fatty acid is produced; and,    f) optionally recovering the ω-3 or ω-6 fatty acid.    
     
     
         14 . A method according to  claim 13  wherein the  Yarrowia  yat1 gene is isolated from  Yarrowia lipolytica.    
     
     
         15 . A method according to  claim 13  wherein the promoter region of the yat1 gene is contained within a nucleic acid molecule as set forth in SEQ ID NO:3.  
     
     
         16 . A method according to  claim 13  wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases and elongases.  
     
     
         17 . A method according to  claim 16  wherein the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ8 desaturase and Δ4 desaturase.  
     
     
         18 . A method according to  claim 13  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.    
     
     
         19 . A method according to  claim 18  wherein the oleaginous yeast is  Yarrowia lipolytica.    
     
     
         20 . A method according to  claim 19 , 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 and  Yarrowia lipolytica  LGAM S(7)1.  
     
     
         21 . A method according to  claim 13  wherein the ω-3 or ω-6 fatty acid is selected from the group consisting of: linoleic acid, α-linolenic acid, γ-linolenic acid, stearidonic acid, dihomo-γ-linoleic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, eicosadienoic acid and eicosatrienoic acid.  
     
     
         22 . A method according to  claim 13  wherein the conditions whereby the at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is expressed are those of nitrogen limitation.  
     
     
         23 . A method according to  claim 22  wherein the conditions of nitrogen-limitation are conditions that induce oleaginy in the transformed oleaginous yeast.