Compositions and methods for succinate production

Abstract

The present application provides genetically modified yeast cell comprising an active succinate fermentation pathway, as well as methods of using these cells to produce succinate.

1 .- 20 . (canceled) 
     
     
         21 . A genetically modified yeast cell of the species  Issatchenkia, Candida  having an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the cells comprise an exogenous succinate exporter gene that produces an enzyme that catalyzes export of succinate from inside the cell to the extracellular environment. 
     
     
         22 . The genetically modified yeast cell of  claim 21 , wherein the active succinate fermentation pathway includes the reactions:
 (a) pyruvate to oxaloacetate;   (b) oxaloacetate to malate;   (c) malate to fumarate; and   (d) fumarate to succinate.   
     
     
         23 . The genetically modified yeast cell of  claim 22 , wherein the cell comprises one or more endogenous genes that encode an enzyme that catalyzes any of reactions (a) though (d). 
     
     
         24 . The genetically modified yeast cell of  claim 23 , wherein the cell comprises one or more copies of one or more endogenous genes selected from the group consisting of pyruvate carboxylase, malate dehydrogenase, fumarase, and fumarate reductase genes. 
     
     
         25 . The genetically modified yeast cell of  claim 24 , wherein one or more of said endogenous genes are operatively linked to an exogenous regulatory element selected from the group consisting of an exogenous promoter and an exogenous terminator. 
     
     
         26 . The genetically modified yeast cell of  claim 22 , wherein the cell comprises one or more exogenous genes that encode an enzyme that catalyzes any of reactions (a) through (d). 
     
     
         27 . The genetically modified yeast cell of  claim 26 , wherein the cell comprises one or more copies of one or more exogenous genes selected from the group consisting of pyruvate carboxylase, malate dehydrogenase, fumarase, and fumarate reductase genes. 
     
     
         28 . The genetically modified yeast cell of  claim 27 , wherein said exogenous pyruvate carboxylase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. 
     
     
         29 . The genetically modified yeast cell of  claim 27  wherein said exogenous malate dehydrogenase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:168, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24. 
     
     
         30 . The genetically modified yeast cell of  claim 27 , wherein said exogenous malate dehydrogenase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:170. 
     
     
         31 . The genetically modified yeast cell of  claim 27 , wherein said exogenous malate dehydrogenase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:172. 
     
     
         32 . The genetically modified yeast cell of  claim 27 , wherein said exogenous fumarase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         33 . The genetically modified yeast cell of  claim 27 , wherein said exogenous fumarate reductase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. 
     
     
         34 . The genetically modified yeast cell of  claim 27 , wherein said exogenous fumarate reductase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, or SEQ ID NO:180. 
     
     
         35 . The genetically modified yeast cell of  claim 21 , wherein said exogenous succinate exporter gene is derived from a fungal succinate exporter source gene and encodes a polypeptide with at least 95% sequence identity to a polypeptide encoded by the fungal succinate exporter source gene. 
     
     
         36 . The genetically modified yeast cell of  claim 31 , wherein said exogenous succinate exporter gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:182 or SEQ ID NO:184. 
     
     
         37 . The genetically modified yeast cell of  claim 22 , wherein the cell comprises a deletion or disruption of an endogenous pyruvate carboxykinase gene. 
     
     
         38 . The genetically modified yeast cell of  claim 22 , wherein the cell comprises a deletion or disruption of an endogenous pyruvate decarboxylase gene. 
     
     
         39 . The genetically modified yeast cell of  claim 21 , wherein the yeast cell is succinate resistant. 
     
     
         40 . A method of producing poly-butylene succinate, the method comprising:
 (a) obtaining succinate produced by culturing the genetically modified yeast cell of  claim 21  in the presence of at least one carbon source;   (b) isolating succinate from the culture; and   (c) converting the succinate to poly-butylene succinate.