Methods for Improved C4-Dicarboxylic Acid Production in Filamentous Fungi

Abstract

The present invention relates to methods of producing C4-dicarboxylic acids, such as malic acid, comprising: (a) cultivating a host cell comprising a polynucleotide encoding a C4-dicarboxylic acid transporter; and (b) recovering the C4-dicarboxylic acid. The present invention also relates to methods for increasing C4-dicarboxylic acid production, as well as host cells comprising the polynucleotides.

What is claimed is: 
     
         1 : A host cell comprising a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter, wherein the heterologous polynucleotide:
 (a) encodes a C4-dicarboxylic acid transporter having at least 95% sequence identity to SEQ ID NO: 4;   (b) hybridizes under high stringency conditions with the full-length complementary strand of SEQ ID NO: 3; or   (c) has at least 95% sequence identity to SEQ ID NO: 3;   wherein the host cell is capable of secreting increased levels of C4-dicarboxylic acid compared to the host cell without the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter when cultivated under the same conditions.   
     
     
         2 : The host cell of  claim 1 , wherein the heterologous polynucleotide encodes a C4-dicarboxylic acid transporter having at least 95% sequence identity to SEQ ID NO: 4. 
     
     
         3 : The host cell of  claim 1 , wherein the heterologous polynucleotide encodes a C4-dicarboxylic acid transporter having at least 97% sequence identity to SEQ ID NO: 4. 
     
     
         4 : The host cell of  claim 1 , wherein the heterologous polynucleotide encodes a C4-dicarboxylic acid transporter having at least 99% sequence identity to SEQ ID NO: 4. 
     
     
         5 : The host cell of  claim 1 , wherein the heterologous polynucleotide encodes a C4-dicarboxylic acid transporter that comprises or consists of SEQ ID NO: 4. 
     
     
         6 : The host cell of  claim 1 , wherein the heterologous polynucleotide hybridizes under high stringency conditions with the full-length complementary strand of SEQ ID NO: 3. 
     
     
         7 : The host cell of  claim 1 , wherein the heterologous polynucleotide hybridizes under very high stringency conditions with the full-length complementary strand of SEQ ID NO: 3. 
     
     
         8 : The host cell of  claim 1 , wherein the heterologous polynucleotide has at least 95% sequence identity to SEQ ID NO: 3. 
     
     
         9 : The host cell of  claim 1 , wherein the heterologous polynucleotide has at least 97% sequence identity to SEQ ID NO: 3. 
     
     
         10 : The host cell of  claim 1 , wherein the heterologous polynucleotide has at least 99% sequence identity to SEQ ID NO: 3. 
     
     
         11 : The host cell of  claim 1 , wherein the heterologous polynucleotide comprises or consists of SEQ ID NO: 3. 
     
     
         12 : The host cell of  claim 1 , wherein the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter is operably linked to a promoter foreign to the polynucleotide. 
     
     
         13 : The host cell of  claim 1 , wherein the host cell is a filamentous fungal host cell. 
     
     
         14 : The host cell of  claim 13 , wherein the host cell is an  Aspergillus  host cell. 
     
     
         15 : The host cell of  claim 14 , wherein the  Aspergillus  host cell is an  Aspergillus oryzae  host cell. 
     
     
         16 : The host cell of  claim 1 , wherein the host cell is capable of secreting at least 50% more C4-dicarboxylic acid compared to the host cell without the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter when cultivated under the same conditions. 
     
     
         17 : The host cell of  claim 1 , further comprising a heterologous polynucleotide encoding a malate dehydrogenase. 
     
     
         18 : The host cell of  claim 1 , further comprising a heterologous polynucleotide encoding a pyruvate carboxylase. 
     
     
         19 : The host cell of  claim 1 , wherein the C4-dicarboxylic acid is malic acid. 
     
     
         20 : A method of producing a C4-dicarboxylic acid, comprising:
 (a) cultivating the host cell of  claim 1  in a medium; and   (b) recovering the C4-dicarboxylic acid.   
     
     
         21 : The method of  claim 20 , wherein the heterologous polynucleotide encodes a C4-dicarboxylic acid transporter having at least 95% sequence identity to SEQ ID NO: 4. 
     
     
         22 : The method of  claim 20 , wherein the C4 dicarboxylic acid is malic acid. 
     
     
         23 : The method of  claim 20 , wherein the host cell is an  Aspergillus  host cell.