Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase promoters for gene expression in oleaginous yeast

Abstract

The promoter regions associated with the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate mutase (gpm) genes have been found to be particularly effective for the expression of heterologous genes in oleaginous yeast. The promoter regions of the invention have been shown to drive high-level 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 cell comprising:
 a) providing a transformed yeast cell having a chimeric gene comprising:
 (i) a promoter region of a gpd  Yarrowia  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   b) growing the transformed yeast cell of step (a) under conditions whereby the chimeric gene of step (a) is expressed.   
     
     
         2 . A method according to  claim 1  wherein the  Yarrowia  gene is isolated from  Yarrowia lipolytica.    
     
     
         3 - 4 . (canceled) 
     
     
         5 . A method according to  claim 1  wherein the promoter region contains at least one mutation that does not diminish its promoter activity. 
     
     
         6 . A method according to  claim 1  wherein the promoter activity is at least about 20% to at least about 400% of the promoter activity of the wildtype promoter activity. 
     
     
         7 . A method according to  claim 1  wherein the transformed yeast cell 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 . (canceled) 
     
     
         10 . A method according to  claim 1  wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases, elongases, 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 for the production of an ω-3 or an ω-6 fatty acid comprising:
 (a) providing a transformed oleaginous yeast comprising a chimeric gene, comprising:
 (i) a promoter region of a . . .  Yarrowia  gene 
 (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; and   (b) contacting the transformed oleaginous yeast of step (a) under conditions whereby the at least one enzyme of the ω-3ω-6 fatty acid biosynthetic pathway is expressed and a ω-3 or ω-6 fatty acid is produced; and   (c) optionally recovering the ω-3 or ω-6 fatty acid.   
     
     
         12 . A method according to  claim 11  wherein the  Yarrowia  gene is isolated from  Yarrowia lipolytica.    
     
     
         13 - 14 . (canceled) 
     
     
         15 . A method according to  claim 11  wherein the coding region of interest encodes a polypeptide selected from the group consisting of: desaturases and elongases. 
     
     
         16 . A method according to  claim 15  wherein the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase and Δ4 desaturase. 
     
     
         17 . A method according to  claim 11  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.    
     
     
         18 . A method according to  claim 17  wherein the oleaginous yeast is  Yarrowia lipolytica.    
     
     
         19 . (canceled) 
     
     
         20 . A method according to  claim 11  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 and docosahexaenoic acid. 
     
     
         21 - 22 . (canceled)