Engineered yeast cells and uses thereof

Abstract

The present application provides engineered yeast cells and uses thereof. In specific embodiments, the yeast cells have a mutation in the GAL2 gene. In specific embodiments, the yeast cells can be used for producing a protein or compound of interest.

We claim: 
     
         1 . A method for regulating the production of a protein of interest in yeast, comprising:
 (i) providing a yeast cell with a mutation in the GAL2 gene, wherein the cell contains a nucleic acid encoding a protein of interest operably linked to a GAL-responsive promoter; and,   (ii) contacting the cell with a molecule which the GAL-responsive promoter responds to and alters the expression of the nucleic acid,   wherein contacting the cell with a greater amount of the molecule leads to a greater alteration of expression of the nucleic acid, thereby regulating the production of the protein of interest.   
     
     
         2 . The method of  claim 1 , wherein the mutation in the GAL2 gene results in the removal of GAL2from the GAL network in the yeast cell. 
     
     
         3 . The method of  claim 1 , wherein the GAL2 gene is operably linked to a promoter selected from the group consisting of: a CUP promoter, a MET promoter, a PHO promoter, and a LAC promoter. 
     
     
         4 . The method of  claim 1 , wherein the yeast cell further comprises a mutation in the GAL1 gene. 
     
     
         5 . The method of  claim 4 , wherein mutation in the GAL1 gene results in the removal of GAL1 from the GAL network in the yeast cell. 
     
     
         6 . The method of  claim 4 , wherein the GAL1 gene is operably linked to a promoter selected from the group consisting of: a CUP promoter, a MET promoter, a PHO promoter, and a LAC promoter. 
     
     
         7 . The method of  claim 1 , wherein the molecule is a galactose or a galactose analog that binds GAL3. 
     
     
         8 . The method of  claim 1 , wherein the GAL-responsive promoter responds to the molecule and increases the expression of the nucleic acid. 
     
     
         9 . The method of  claim 1 , wherein the GAL-responsive promoter responds to the molecule and decreases the expression of the nucleic acid. 
     
     
         10 . The method of  claim 9 , wherein the yeast cell further comprises a mutation in one or more genes selected from the group consisting of: GAL4, GAL80, and GAL3. 
     
     
         11 . The method of  claim 1 , wherein the GAL-responsive promoter is a native promoter. 
     
     
         12 . The method of  claim 1 , wherein the GAL-responsive promoter is selected from the group consisting of: the GAL1 promoter, GAL2 promoter, GAL3 promoter, GAL4 promoter, GAL80 promoter, and GAL1,10 promoter. 
     
     
         13 . The method of  claim 1 , wherein the GAL-responsive promoter is a heterologous promoter or a promoter that is not a naturally-occurring yeast promoter. 
     
     
         14 . The method of  claim 1 , wherein the GAL-responsive promoter comprises one or more GAL4 upstream activation sequences. 
     
     
         15 . The method of  claim 1 , wherein the GAL-responsive promoter is a synthetic promoter comprising one or more GAL4 upstream activation sequences. 
     
     
         16 . The method of  claim 15 , wherein the synthetic promoter further comprises a constitutive promoter linked with the one or more GAL4 upstream activation sequences. 
     
     
         17 . The method of  claim 16 , wherein the constitutive promoter is activated in response to the molecule. 
     
     
         18 . The method of  claim 16 , wherein the constitutive promoter is repressed in response to the molecule. 
     
     
         19 . A method for regulating the production of a compound of interest in yeast, comprising:
 (i) providing a yeast cell with a mutation in the GAL2 gene, wherein the cell contains a nucleic acid encoding a protein operably linked to a GAL-responsive promoter, wherein the protein regulates the production of a compound of interest; and,   (ii) contacting the cell with a molecule that the promoter responds to and increases the expression of the nucleic acid,   wherein contacting the cell with a greater amount of the molecule leads to a greater amount of expression of the nucleic acid, thereby regulating the production of the compound of interest.   
     
     
         20 . A method for regulating the production of a compound of interest in yeast, comprising:
 (i) providing a cell with a mutation in one or more genes selected from the group consisting of: GAL2, GAL4, GAL80, and GAL3, wherein the cell contains a nucleic acid encoding a protein operably linked to an inducible promoter, wherein the protein regulates the production of a compound of interest; and,   (ii) contacting the cell with a molecule that the promoter responds to and decreases the expression of the nucleic acid,   wherein contacting the cell with a greater amount of the molecule leads to a lower amount of expression of the nucleic acid, thereby regulating the production of the compound of interest.   
     
     
         21 . A yeast cell, comprising:
 (i) a mutation in the GAL2 gene; and,   (ii) a nucleic acid encoding a protein of interest operably linked to a GAL-responsive promoter, wherein the mutation alters the production of the protein of interest in response to galactose or an analog thereof as compared the production of the protein of interest with the nucleic acid in a corresponding wild type yeast cell.   
     
     
         22 . The yeast cell of  claim 21  further comprising a mutation in the GAL1 gene. 
     
     
         23 . A yeast cell, comprising:
 (i) a mutation in the GAL2 gene; and,   (ii) a nucleic acid encoding a protein operably linked to a GAL-responsive promoter, wherein the protein regulates a compound of interest, and wherein the mutation alters the production of the protein of interest in response to galacotose or an analog thereof as compared the production of the protein of interest with the nucleic acid in a corresponding wild type yeast cell.   
     
     
         24 . The yeast cell of  claim 23  further comprising a mutation in the GAL1 gene.